Methods and devices for standoff differential Raman spectroscopy with increased eye safety and decreased risk of explosion

ABSTRACT

A compact, portable Raman spectrometer makes fast, sensitive standoff measurements at little to no risk of eye injury or igniting the materials being probed. This spectrometer uses differential Raman spectroscopy and ambient light measurements to measure point-and-shoot Raman signatures of dark or highly fluorescent materials at distances of 1 cm to 10 m or more. It scans the Raman pump beam(s) across the sample to reduce the risk of unduly heating or igniting the sample. Beam scanning also transforms the spectrometer into an instrument with a lower effective safety classification, reducing the risk of eye injury. The spectrometer&#39;s long standoff range automatic focusing make it easier to identify chemicals through clear and translucent obstacles, such as flow tubes, windows, and containers. And the spectrometer&#39;s components are light and small enough to be packaged in a handheld housing or housing suitable for a small robot to carry.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a bypass continuation of International ApplicationNo. PCT/US2019/021312, filed Mar. 8, 2019, and entitled “Methods andDevices for Standoff Differential Raman Spectroscopy with Increased EyeSafety and Decreased Risk of Explosion,” which in turn claims thepriority benefit, under 35 U.S.C. 119(e), of: U.S. ProvisionalApplication No. 62/793,601 entitled “EYE-SAFE AND EXPLOSION MITIGATEDSTANDOFF DIFFERENTIAL RAMAN SPECTROSCOPY,” filed Jan. 17, 2019; U.S.Provisional Application No. 62/692,657 entitled “EYE-SAFE AND EXPLOSIONMITIGATED STANDOFF DIFFERENTIAL RAMAN SPECTROSCOPY,” filed Jun. 29,2018; and U.S. Provisional Application No. 62/678,065 entitled “DEVICEAND METHODS FOR EYE-SAFE AND EXPLOSION MITIGATED STANDOFF RAMANSPECTROSCOPY,” filed May 30, 2018. Each of these applications isincorporated herein by reference in its entirety. This application alsodirectly claims the priority benefit, under 35 U.S.C. 119(e), of: U.S.Provisional Application No. 62/793,601 entitled “EYE-SAFE AND EXPLOSIONMITIGATED STANDOFF DIFFERENTIAL RAMAN SPECTROSCOPY,” filed Jan. 17,2019; U.S. Provisional Application No. 62/692,657 entitled “EYE-SAFE ANDEXPLOSION MITIGATED STANDOFF DIFFERENTIAL RAMAN SPECTROSCOPY,” filedJun. 29, 2018; and U.S. Provisional Application No. 62/678,065 entitled“DEVICE AND METHODS FOR EYE-SAFE AND EXPLOSION MITIGATED STANDOFF RAMANSPECTROSCOPY,” filed May 30, 2018. As noted above, each of theseapplications is incorporated herein by reference in its entirety.

BACKGROUND

Raman spectroscopy has proven to be a highly valuable portableanalytical tool for accurate chemical identifications. In the mostcommon implementation, a Raman spectrometer requires the operator tohold the device close (e.g., less than couple of centimeters away) tothe substance under investigation, illuminating the substance with apump laser and collecting the Raman scattering. With typical portableRaman instruments, the laser hazard zone is half a meter or larger andlaser training is required for the operators of such a tool since it istypically classified as a Class 3B instrument. Standoff Raman systemswith a meter or more of standoff distance from sample also exist but thelaser hazard zone is much larger (e.g., a few meters) preventing easyadoption of such systems in the field in addition to burdening theoperators and manufacturers with a more onerous laser safety trainingclass.

For visible or near-infrared (NIR) Raman systems laser hazard is due toocular damage, in particular thermal damage to the retina, from thelaser used as the Raman pump. The damage is a function of radiance(W/m²/sr), distance of Raman laser source to the eye, wavelength, andexposure time. For a standoff Raman system, the laser power exits thesystem output aperture and converges to a spot outside the system, on atarget sample.

SUMMARY

Examples of the devices and methods described here aim at increasing ormaximizing the allowable output laser power of an instrument of acertain laser class, while also mitigating risks (e.g., explosion) orother issues (e.g., perturbation of the kinetics of a reaction) linkedto local heating of the analyte. A spectroscopic laser-based instrumentwith an assigned class of 3B may be downgraded to a class of 3R (or,similarly, an instrument with an assigned class of 3R may be downgradedto class 1) with the use of the devices and methods presented here. Aspectroscopic laser-based instrument that can ignite some analytes maybe made to reduce or eliminate the risk of igniting these analytes usingthe devices and methods presented here. A spectroscopic laser-basedinstrument that causes significant local heating of an analyte may bemade to cause nominal local heating of the analyte using the devices andmethods presented here.

Here we present a method for implementing an eye-safe (Class 3R orlower) Raman system using beam scanning. The system operates at astandoff distance of a few centimeters to a few meters with a laseroutput power that would correspond to a higher-class system (from an eyesafety point of view) if beam scanning was not used (e.g., the systemwould be class 3B without scanning and can be made as safe as a class 3Rinstrument with proper beam scanning).

The signal to noise ratio (SNR) of a given Raman system increases as theRaman pump power increases. There is thus a competition between thedesire for a high SNR system, providing rapid spectral acquisition forfast chemical analysis, and the desire for an eye-safe system. A secondtrade-off is with regard to the etendue of the spectrometer: aspectrometer with a large etendue can stare at a wider area on thesample. This is often used to improve eye safety by widening the Ramanpump spot size on the sample, thereby reducing the radiance. However,increasing the etendue of the spectrometer generally reduces itsresolution. We present here a system that allows to maintain a smalletendue (e.g. smaller than 0.2 mm²) and corresponding potential for highresolution, together with a high Raman pump power (e.g. larger than 5mW) for good SNR and fast spectral acquisition, while reducing the risksof ocular damage.

A high radiance on the sample is also sometimes associated withexplosion or ignition risks of the material analyzed. Alternatively, thesample may heat up so much under the high radiance of the Raman pumpthat it emits an incandescent glow that interfere with or altogetherprevent the measurement. From this point of view, there is also a needto reduce the radiance on the sample, which is addressed by the systempresented here.

If the laser spot is scanned on a sample over a scan area with a highenough speed, the effective ocular laser spot size approaches that ofthe scan area, significantly increasing the threshold for eye-safe laseroperation. (The scan is an areal scan that probes a finite volume orlayer of material since the beam has a finite depth of focus and thereis scattering inside the sample.) Whether this effective average exposedarea or the instantaneous scanned spot should be considered depends onthe exact scan parameters and may be evaluated as outlined in the lasersafety standards. The instantaneous spot size, scan area, and scanspeed, as well as the divergence of the laser beam can be optimized orselected to produce such enhanced eye-safe standoff Raman system. Inparticular the scan pattern can be optimized for homogeneousillumination to prevent “hot regions” in the scan area. If the laserspot dwells for too long in a region, the ocular damage threshold can bereached, which means the irradiance on the retina can exceed thethreshold for damage, for example thermal damage. Empty or hollow scanpatterns can have small effective scan areas. For example, a fastcircumferential scan produces an effective scan area which isapproximately the product of diameter of laser spot and thecircumference of the scanned circle. If the inner area of the circle isscanned as well, the total scan area (and hence the effective spot size)may approach the area of the circle, which is larger than the effectivearea of the circular scan.

By properly choosing the scan parameters, as explained below, to producelarge effective area for ocular damage, the temperature rise on thesample surface is also reduced. Both ocular damage and sampletemperature rise are due to concentrated laser power dwelling forsufficient time on the same spot. The thermal diffusion of retinaltissue would likely be different than the thermal diffusion of thesample but in both cases a reduced temperature rise can be obtained withthe use of appropriate scanning.

Eye-safe lasers are typically understood to be lasers that emit lessthan 5 mW of output power (e.g., class 1, 2 and 3R lasers, but not class3B lasers), Eye-safe lasers can be operated without special safetytraining. Class 3R instruments are considered eye-safe when handledcarefully and have a small hazard potential for accidental exposure.Some commercial laser pointers are class 3R instruments.

More generally, the devices and methods presented here lower the eyesafety classification of a given system using laser beam scanning. Forexample, a class 3B system can be transformed into a class 3R systemusing laser scanning. Or a Class 3R system may be transformed into aClass 1 system. Or a Class 4 system may be transformed into a Class 3Bsystem.

While the inventive devices and methods are presented here in thecontext of Raman spectroscopy, implying the use of a pump laserilluminating a sample, and whereby the scattered light is collected byan optical system and focused into a spectrometer entrance slit, theinventive devices and methods are applicable to other laser spectroscopytechniques since they address in general increasing eye safety forinstruments requiring the illumination of a sample with a bright focusedbeam of light. Other forms of Raman spectroscopy (coherent Ramanspectroscopy, resonant Raman spectroscopy, difference Ramanspectroscopy, spatially offset Raman spectroscopy, surface-enhancedRaman spectroscopy, etc.) can benefit similarly from the devices andmethods described here as well as other laser-based techniques, such aslaser induced fluorescence spectroscopy.

The devices and methods presented here are applicable to laser-basedinstruments in general. They can be implemented without an enclosure orshield to prevent the beam from exiting a certain perimeter. They are ofparticular utility for standoff instruments with a certain length ofopen air (e.g., >2 cm) between the instrument and the sample. Thetechnology presented here involves laser beam scanning, together withthe implementation of fail-safe monitoring and automatic shut-offmechanisms. It allows the use of higher power laser than would beotherwise allowed without laser beam scanning, within a certain laserclass.

An inventive Raman system may have a standoff distance larger than 2centimeters. The system includes several features designed to mitigatethe unique challenges due to the increased standoff distance, and theinterference of ambient light and the signal variations resulting fromthe operator's hand movements, as well as challenges common to mostRaman systems, such as sample fluorescence, risk of sample explosion orignition, and eye safety risks.

An example Raman system can be implemented as a handheld standoffinstrument, a Raman microscope, or a general chemical analysis tool, forexample, used to monitor chemical reactions, identify materials, confirma material composition, or quantify relative concentrations in a mixtureof several materials. It may have one or more of the following features:

-   -   The system may measure a sample located at a distance greater        than 2 centimeters from the instrument;    -   The system may allow for handheld operation and thus assume a        dynamic signal, within particular variations in ambient light        background, sample fluorescent background, as well as Raman        signal during the measurement;    -   The system may use at least two Raman lasers in a difference        Raman mode of operation to mitigate the effects of sample (or        substrate) fluorescence. A tunable laser may be used instead of        several lasers since the spectra are acquired successively with        different Raman pump wavelength;    -   The system may also use difference Raman technology to remove        the background spectrum due to blackbody emission from a hot        sample;    -   The laser or lasers used as Raman pump laser may be uncooled,        allowing to reduce the system weight and power consumption. A        small amount of the Raman laser light may be leaked in the Raman        probe towards the spectrometer, causing the zero-Stokes shift        line (Rayleigh scattering) to be visible in the Raman spectrum.        The zero-Stokes shift line provides a reference to compute the        Stokes shifts even if the Raman laser wavelength varies over        time;    -   The system may use short integration frame times with        interleaved Raman frames and background frames to provide        robustness against signal variations;    -   The system may be compatible with laser beam scanning, which        increases eye safety of the instrument and reduces the risk of        sample ignition or explosion for a given laser power output and        spot size;    -   The timing of the different processes (Raman laser modulation or        switching, spectral acquisition, laser beam scanning) may be        synchronized and chosen in a manner aimed at reducing or        minimizing measurement fluctuations due to laser beam scanning        or potential ambient light modulation;    -   The system may include a camera imaging the sample;    -   The system may use the visual information from the camera for        image stabilization;    -   The system may allow the user to indicate a feature of interest,        for example, by pressing a touchscreen displaying an image of        the area surrounding a sample, to indicate a desired measurement        spot. The system can lock onto this spot, e.g., by automatically        steering the telescope optics to focus the Raman probe to that        spot and maintaining that aim. The system maintains this aim by        correcting automatically for the hand movements of the operator        using information from the camera.    -   The system may have an adjustable focus distance;    -   The system may have an autofocus system to automatically adjust        the focus distance and increase or maximize Raman signal;    -   The auto-focus system may allow for an offset setting so that        the system may be focused a short distance away from a first        scattering surface;    -   The auto-focus may be able to detect multiple interfaces along        the optical axis and sequentially tune the instrument focus to        realize measurements at or near these interfaces, or at any        point between consecutive interfaces.

Unless noted otherwise, the laser safety considerations presented hereare derived from the 2014 revision of the IEC 60825-1 standard since USFederal Laser Notice 50 allows classification according to IEC 60825-1,instead of compliance with 21 CFR 1040.10 and 1040.11. However, sincethese eye-safety standards refer to the same bio-physical constraints,they tend to present similar derivations and the strategy presented hereis applicable in general to increase eye-safety of a laser-basedinstrument. Details of some bio-physical considerations underlying theeye-safety standards can be found in Francois C. Delori, Robert H. Webb,and David H. Sliney, “Maximum permissible exposures for ocular safety(ANSI 2000), with emphasis on ophthalmic devices,” J. Opt. Soc. Am. A24, 1250-1265 (2007), which is incorporated herein by reference in itsentirety.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. All combinations ofclaimed subject matter appearing at the end of this disclosure arecontemplated as being part of the inventive subject matter disclosedherein. It should also be appreciated that terminology explicitlyemployed herein that also may appear in any disclosure incorporated byreference should be accorded a meaning most consistent with theparticular concepts disclosed herein.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is an example of scanning standoff Raman system.

FIGS. 2A-2C illustrates different possible scan patterns. A lowfill-factor scan pattern, such as the circumferential pattern shown inFIG. 2A would heat selected portions of the surface of the sample or theretina of the eye more than the higher fill-factor patterns shown inFIG. 2B (raster pattern) and FIG. 2C (Lissajous pattern).

FIG. 3A is a schematic of an example beam scanning device.

FIG. 3B is a schematic of an example of a retracing beam scanningdevice.

FIG. 4A is a schematic of a mirror monitoring device.

FIG. 4B illustrates time signals (upper and lower plots) from afour-quadrant detector.

FIG. 5 is a schematic of an example fail-safe mechanism.

FIG. 6 illustrates a process for determining system design parametersthat can be adjusted to obtain a desired laser classification for thesystem.

FIG. 7 introduces the notations used to characterize the system etendue.

FIG. 8A is a schematic of a standoff sample temperature monitoringsystem.

FIG. 8B is a schematic of another embodiment of a standoff temperaturemonitoring system.

FIG. 9 is a schematic of a standoff Raman system's fail-safe mechanism(e.g., for explosion or ignition risk mitigation) based on measurementof the sample temperature.

FIG. 10 illustrates the different Raman signals that can be observed andmay be useful for temperature measurement.

FIG. 11 illustrates the use of a Raman marker for temperaturemeasurement of the analyte.

FIGS. 12A-12E is a collection of schematics representing differentmeasurement conditions for a standoff Raman analyzer, or otherlaser-based, standoff spectrometers, including standoff measurements in:FIG. 12A—a solid sample; FIG. 12B—a liquid in a container through thecontainer wall; FIG. 12C—a liquid in a container through a containeropening; FIG. 12D—an analyte in a reactor; and FIG. 12E—an analyte in aflow cell.

FIG. 13A is a photograph of a handheld Raman standoff differentialspectroscopy system.

FIG. 13B illustrates a handheld Raman standoff differential spectroscopysystem in a measurement setting.

FIG. 14 is a block diagram of an example handheld differential Ramanspectroscopy system.

FIG. 15 is a block diagram of a handheld differential Raman spectroscopysystem with an optical switch that switches between Raman pump lasersthat emit at slightly different wavelengths.

FIG. 16 shows a two-part differential Raman spectroscopy system with aprobe module connected to a core module via electrical cable(s) andoptical fiber(s).

FIG. 17 illustrates the acquisition cycle for differential Ramanspectroscopy with frame periods selected based on beam scanning for eyesafety/explosion mitigation and ambient light modulation.

FIGS. 18A and 18B illustrate the use of visible laser beams to mark theRaman pump beam's focus in a Raman spectroscopy system, with FIG. 18Aillustrating a first position of the movable lens, and FIG. 18Billustrates a second position of the movable lens that is different thanthe first position.

FIG. 19 illustrates the use of a camera system for imaging and trackinga standoff Raman system's aim point.

FIG. 20 illustrates focusing a Raman pump beam through obstructions(e.g., a window and a beaker wall) with a handheld standoff differentialRaman spectroscopy system.

FIG. 21 illustrates a two-part standoff differential Raman spectroscopysystem used with a robot.

FIG. 22 illustrates a two-part standoff differential Raman spectroscopysystem used in a laboratory to measure a sample in or on a slide.

FIG. 23 illustrates a two-part standoff differential Raman spectroscopysystem used in a laboratory to measure a liquid sample in a glasscontainer.

FIG. 24 illustrates a two-part standoff differential Raman spectroscopysystem controlled with a wireless controller, with a core module and aprobe module mounted on a fixed ring holder to the side of a sample in abeaker.

FIGS. 25A and 25B illustrate a two-part standoff differential Ramanspectroscopy system measuring, from above, a first workingdistance/depth in a liquid sample and a second working distance/depth inthe liquid sample, respectively.

FIGS. 26A and 26B illustrate a two-part standoff differential Ramanspectroscopy system measuring, through the sidewall of a container, afirst position in a liquid sample and a second position in the liquidsample, respectively.

FIG. 27 illustrates a modular standoff differential Raman spectroscopysystem.

FIG. 28A illustrates a stand-alone Raman macroscope system.

FIG. 28B illustrates a tripod-mounted Raman macroscope system.

FIG. 28C illustrates a Raman macroscope or stereo-microscope mount withincluded height adjustment.

FIG. 29 illustrates a possible result screen.

FIG. 30 illustrates several features of the Raman macroscope system.

FIG. 31 illustrates a diagram of the Raman macroscope system.

FIG. 32 illustrates a Raman macroscope system similar to the one shownin FIG. 31 with a motorized sample stage.

FIG. 33 illustrates a Raman macroscope system similar the one shown inFIG. 31 with the motion controller controlling movement of the entiresystem.

FIG. 34 illustrates acquisition of polarized difference Ramaninformation/spectra.

FIG. 35A is a front perspective photograph of a standoff Raman systemmounted on a microscope stage.

FIG. 35B is a photograph of the standoff Raman system of FIG. 35A duringtypical use.

FIG. 36 is a photograph of a standoff Raman system used to monitor areaction inside a vessel.

FIG. 37 is a photograph of a handheld standoff Raman system identifyinga chemical compound through multiple obstructions.

FIGS. 38A and 38B illustrate a reaction vessel that is designed toprevent formation of condensation on its walls and that includes acavity for circulation. FIG. 38A illustrates the cavity as closed. FIG.38B illustrates the cavity as open and permitting circulation of afluid.

FIGS. 39A and 39B illustrate a dry enclosure used prevent formation ofcondensation on the walls of a reaction vessel. FIG. 39A illustrates theRaman system outside the dry enclosure.

FIG. 39B illustrates the Raman system inside the dry enclosure.

FIG. 40 illustrates a dry gas flow used prevent formation ofcondensation on the walls of a reaction vessel.

The skilled artisan will understand that the drawings primarily are forillustrative purposes and are not intended to limit the scope of theinventive subject matter described herein. The drawings are notnecessarily to scale; in some instances, various aspects of theinventive subject matter disclosed herein may be shown exaggerated orenlarged in the drawings to facilitate an understanding of differentfeatures. In the drawings, like reference characters generally refer tolike features (e.g., functionally similar and/or structurally similarelements).

DETAILED DESCRIPTION

Eye Safety and Explosion Mitigation

A method of Raman spectroscopy includes projecting a first Raman pumpbeam at a first wavelength onto a sample from a standoff distance of atleast 10 centimeters. The method also includes detecting a first Ramansignal emitted by the sample in response to the first Raman pump beam,the first Raman signal representing a first Raman signature and a firstbackground signature. The method further includes projecting a secondRaman pump beam at a second wavelength different than the firstwavelength onto the sample from the standoff distance. The method alsoincludes detecting a second Raman signal emitted by the sample inresponse to the second Raman pump beam, the second Raman signalrepresenting a second Raman signature and a second background signature.The method further includes generating a post-processed signature basedon at least one of the first Raman signal or the second Raman signal,the post-processed signature having a background lower than or equal tothe first background signature.

A method of Raman spectroscopy includes acquiring interleavedmeasurements of a first Raman signal of a sample using a Raman pump at afirst wavelength, a second Raman signal of the sample using a Raman pumpat a second wavelength different than the first wavelength, and ambientlight transmitted and/or scattered by the sample from a distance of atleast 2 cm from the sample. The method further includes generating apost-processed signature based on the interleaved measurements of thefirst Raman signal, the second Raman signal, and the ambient light.

A method of Raman spectroscopy of a sample includes scanning a firstRaman pump beam at a first wavelength in a scan pattern across a surfaceof sample over a first scan period and measuring a first Raman signalscattered by the sample in response to the first Raman pump beam over afirst integration period. The method also includes scanning a secondRaman pump beam at a second wavelength different than the firstwavelength in the scan pattern across the surface of sample over asecond scan period and measuring a second Raman signal scattered by thesample in response to the second Raman pump beam over a secondintegration period. The method further includes measuring ambient lighttransmitted and/or scattered by the sample over a third integrationperiod, and forming a post-processed signature based on the first Ramansignal, the second Raman signal, and the ambient light.

A method of Raman spectroscopy of a sample can include measuring a rangeof between about 2 centimeters and 400 centimeters from a Ramanspectroscopy system to the sample, and automatically focusing a Ramanpump beam emitted by the Raman spectroscopy system based on the rangefrom the Raman spectroscopy system to the sample. The method can alsoinclude detecting a Raman signal from the sample in response to theRaman pump beam, and estimating a Raman signature of the sample based onthe Raman signal.

A method of Raman spectroscopy of a sample includes illuminating thesample, via a telescope, with a first ranging beam propagating along anoptical axis of the telescope to form a first spot on the sample. Themethod also includes illuminating the sample, via the telescope, with asecond ranging beam propagating along a marginal ray of the telescope toform a second spot on the sample. The method includes detecting adistance between the first spot and the second spot and adjusting afocus of the telescope based on the distance between the first spot andthe second spot. The method also includes illuminating the sample, viathe telescope, with a Raman pump beam propagating along the optical axisof the telescope and measuring a Raman signal scattered by the sample inresponse to the Raman pump beam.

A Raman spectroscopy system can include a camera to acquire an image ofa scene and a processor, operably coupled to the camera, to identify atarget in the scene based on the image of the scene. The system can alsoinclude a laser, in optical communication with a sample in the scene, toilluminate the target with a Raman pump beam. The system furtherincludes a detector, in optical communication with the sample, to detecta Raman signal scattered by the target in response to the Raman pumpbeam.

A system for Raman spectroscopy includes a housing and at least oneranging laser, disposed within the housing, to emit a first visibleranging beam and a second visible ranging beam. The system furtherincludes a telescope, disposed with the housing in optical communicationwith the at least one ranging laser, to project the first visibleranging beam to a first spot on a sample located between about 2centimeters and about 4 meters from the housing and to project thesecond visible ranging beam to a second spot on the sample, the firstspot and the second spot providing an indication of a range to thesample. The system also includes at least one Raman pump laser, disposedwithin the housing, to emit a first Raman pump beam at a firstwavelength and a power level of at least about 10 mW during a firstperiod based on a flicker period of ambient illumination and to emit asecond Raman pump beam at a second wavelength different than the firstwavelength and a power level of at least about 10 mW during a secondperiod equal to and after the first period. The system also includes abeam scanner, disposed within the housing in optical communication withthe at least one Raman pump laser and the telescope, to scan the firstRaman pump beam across a portion of the sample during the first periodand to scan the second Raman pump beam across the portion of the sampleduring the second period, the telescope focusing the first Raman pumpbeam and the second Raman pump beam on or near a surface of the sample.The system also includes a detector, disposed within the housing inoptical communication with the sample, to detect a first Raman signalfrom the sample during the first period, a second Raman signal from thesample during the second period, and the ambient illuminationtransmitted or scattered by the sample during a third period equal toand after the second period. The system further includes a processor,disposed within the housing and operably coupled to the detector, toproduce a post-processed signature based on the first Raman signal, thesecond Raman signal, and the ambient illumination signal, thepost-processed signature having a lower fluorescent background than thefirst Raman signal and the second Raman signal.

A spectroscopy system includes a laser to illuminate a sample at adistance of at least 2 centimeters from the spectroscopy system with alaser beam having first laser safety class, and a beam steering element,in optical communication with the laser, to scan a spot formed by thelaser beam across a surface of a sample. The spectroscopy system alsoincludes a monitoring system, operably coupled to the beam steeringelement and/or to the laser, to detect a malfunction of the beamsteering system that would cause the laser beam to exceed a maximumpermissible exposure for a second laser safety class and to at least oneof attenuate, redirect, block, or turn off the laser beam in response todetection of the malfunction.

A spectroscopy system includes a laser configured to illuminate a sampleat a distance of at least 2 centimeters from the spectroscopy system,and a beam steering element, in optical communication with the laser, toscan a spot formed by the laser beam across a surface of a sample. Thesystem also includes a monitoring system to measure the sampletemperature and automatically shut off the laser or reduce its emissionpower should a temperature rise above a certain threshold be detected.

A spectroscopy system can be configured to illuminate a sample at adistance of at least 10 centimeters from the spectroscopy system with aRaman pump beam at a wavelength of about 700 nanometers to about 1050nanometers and with a power of at least 10 mW

A method of spectroscopy can include emitting a Raman pump beam from alaser, the Raman pump beam having a power of more than about 5milliwatts and a wavelength of about 700 nanometers to about 1050nanometers. The method can also include focusing the Raman pump beam toa spot on a sample at a distance of about 2 centimeters to about 10meters from the laser, scanning the spot across at least a portion ofthe sample, and detecting radiation emitted by the sample in response tothe Raman pump beam.

A spectroscopy system can include a housing, an optical assembly, and acamera, disposed within the housing in optical communication with theoptical assembly, to acquire an image of a sample via the opticalassembly. The spectroscopy system can also include a laser, disposedwithin the housing, to emit a Raman pump beam, and a beam-steeringelement, disposed within the housing in optical communication with thelaser and the optical assembly, to scan the Raman pump across the samplevia the optical assembly. The spectroscopy system can also include aspectrometer, disposed within the housing, to detect Raman lightscattered by the sample in response to the Raman pump beam.

A spectroscopy system includes a housing, an optical assembly, and acamera, disposed within the housing in optical communication with theoptical assembly, to acquire an image of a sample via the opticalassembly. The spectroscopy system also includes a laser, disposed withinthe housing, to emit a Raman pump beam and an actuator to move theoptical assembly with respect to the sample. The spectroscopy systemfurther includes a spectrometer, disposed within the housing, to detectRaman light scattered by the sample in response to the Raman pump beam.

The technology presented here can be used to reduce the laser energyimpinging at any one point of the sample, and, through imaging by thelens of the eye at any one point of the retina, increase eye safety. Weachieve this here by scanning the Raman pump beam over a small area ofthe sample to be analyzed. For the sake of simplicity, theimplementation details and calculations refer to a Raman system with aNIR pump laser, with wavelength between 700 nm and 1050 nm. Otherwavelengths are also possible. The accessible emission limit (AEL) for aClass 3R instrument in this wavelength range and for exposure timesrelevant to Raman analysis is:AEL(in Joules)=3.5×10⁻³ C ₄ C ₆ t ^(0.75)  (1)AEL(in Watt)=3.5×10⁻³ C ₄ C ₆ t ^(−0.25)  (2)where C₄ is a correction factor accounting for the laser wavelength(C₄=10^(0.002(λ-700)), with λ being the laser wavelength in nanometers),C₆ is a correction factor accounting for the source size (extendedsource, C₆=α/α_(min), with a the angular subtended by the apparentsource and α_(min)=1.5 mrad), and t is the exposure duration.

Established laser-safety standards are mostly concerned with fixed(non-scanning) lasers, either pulsed or operated in continuous wave. Thetreatment of scanned beams is described in the IEC 60825-1 Standard asfollows: “For laser products emitting a scanned beam, depending on theaccommodation condition to image the apparent source, a scanning beamcan result in the image of the apparent source being scanned across theretina, resulting in a moving apparent source. If a moving apparentsource is to be accounted for in the classification, the classificationof the product is based on the evaluation method described here forextended sources (in contrast to the simplified analysis where a smallsource is assumed to be stationary). The moving apparent source is to beevaluated as described in 4.3. d) [i.e. non-uniform, non-circular ormultiple apparent sources] with due consideration of the repetitivepulse nature of the accessible emission determined with the respectiveangle of acceptance.” Eye-safety assessment should follow three rules:

-   -   The exposure from any single pulse within a pulse train should        not exceed the maximum permissible exposure (MPE) for a single        pulse.    -   The average exposure for a pulse train of exposure duration T        should not exceed the MPE for a single pulse of exposure        duration T.    -   The exposure per pulse should not exceed the MPE for a single        pulse multiplied by a correction factor (C₅) accounting for        repeated exposures.

In studying a scanned beam, we calculate the average exposure over allor part of the scanned area, for varying time scales including the scanperiod, in order to find the most restrictive condition. Pulsed exposureis used to consider the transient exposure of a spot as the laser beamtravels through it. In this case, we consider a scanned laser to beequivalent to a pulsed laser with a spot size equal to the spot sizeobtained when the laser scanning is turned off, and with a pulseduration equal to the time it takes for the beam to be scanned across alength equal to the spot diameter. For the repeated pulse condition,that pulse is considered repeated every time the scan pattern crossesthat same area.

FIG. 6 illustrates a process for determining the system designparameters that can be adjusted to obtain a desired laser classificationfor any system described herein. At 600, the main design parameters,discussed in greater detail below, are used as inputs to calculate, theexposure levels reaching the eye under several possible exposure (atsteps 601/602/603) and measurement conditions (at step 604), and each ofthe average exposure, pulsed exposure, and repeated pulsed exposureconditions are described in greater detail below. At 604, the case withthe highest exposure is identified and compared with the correspondingaccessible exposure limits, according to published eye safety standards.Based on the result of this comparison, a laser classification can bedetermined, and if desired the parameters can be adjusted at step 606 toadjust the laser classification. Note that the final laserclassification may entail additional requirements, such as safetymarkings or the presence of fail-safe mechanism, warning lights, keylocks, etc., that are not considered here since they can beindependently added as desired.

We detail below some example system parameters (values, ranges, degreesof freedom, etc.) that can affect eye safety of the system underconsideration:

-   -   Laser (Raman pump laser) wavelength: e.g., between about 250 nm        and about 1100 nm.    -   Laser peak output power (as measured at the system output port):        e.g., between about 5 mW and about 5 W.    -   Laser duty cycle: e.g., between 1% and 100%.    -   Laser spot size (without scanning, measured on the sample):        e.g., between about 1 micrometer and about 500 micrometers in        diameter.    -   Beam diameter at the system output port (e.g., the telescope's        exit lens, which may form the boundary between the instrument        and the environment surrounding the instrument): e.g., between        about 6 mm and about 100 mm, depending on the desired standoff        distance.    -   System focal length (distance from beam output port and the        focused spot): e.g., between about 1 cm and about 10 meters.    -   Scan maximum angular deviation (measured at the system output        port): Consistent with a position shift of the focused spot of        less than about 1 inch on the sample, as described in greater        detail herein.    -   Scan speed: full accessible deviation range scanned in a time        (scan period) that is from about 5 microseconds to about 500        milliseconds.    -   Scan pattern, and the fill-factor of the scan pattern and its        uniformity (ratio of highest exposure to average exposure)        within the bound of the scan maximum angular deviation.

Of these parameters, two are particularly significant for the ability ofa Raman system to obtain high quality data, in terms of signal-to-noiseratio and spectral resolution: the laser power and the system etendue.The etendue is a measure of how diffuse a light source is, in area andangle. For a Raman spectrometer, the etendue can be approximated as:

$\begin{matrix}{{G = \frac{A_{laser}A_{lens}}{s^{2}}}\mspace{11mu}} & (3)\end{matrix}$where G is the etendue, A_(laser) is the area of the source (laserfacet, or optical fiber facet), A_(lens) is the illuminated area of thefocusing lens (the telescope used to image the laser or fiber facet onthe sample is simplified to a simple equivalent lens), and s is thedistance between the focusing lens and the laser source. The laser spotdiameter d on sample is directly related to A_(laser) by themagnification provided by the focusing lens:

${d = {\frac{s_{1}}{s}\sqrt{\frac{4}{\pi}A_{laser}}}},$where s₁ is the distance between the focusing lens and the sample(system focal lens). The beam diameter D at the system output is suchthat

$A_{lens} = {\frac{\pi}{4}{D^{2}.}}$The etendue can also be expressed as:G=πA _(spot)(NA)²  (4)where A_(spot) is the area illuminated by the laser on sample (i.e.,

$A_{spot} = {\frac{\pi}{4}d^{2}}$using the notations above), and NA is the system numerical aperture:

${NA} = \frac{D}{2s_{1}}$using the notations introduced above. The notations are also illustratedin FIG. 7, which illustrates a Raman pump source 700 (e.g., a laserfacet or optical fiber facet) with area A_(laser) is imaged by afocusing lens 701 onto a sample 702. The laser spot 703 has an areaA_(spot) while the beam illuminates an area A_(lens) of the focusinglens. The distance between the source and the lens is s while thedistance between the lens and the sample is s₁.

In general, decreasing the laser power and/or increasing the etenduetends to increase eye safety and mitigate explosion risk of the samplebecause doing so decreases the sample irradiance as well as the amountof light that can be collected by the eye pupil. However, decreasing thelaser power reduces the signal-to-noise ratio and consequently increasesthe measurement time, while increasing the etendue tends to reduce thespectral resolution, and can also negatively affect collectionefficiency and thus signal-to-noise ratio. The devices and methodspresented here increase eye safety while maintaining a high laser powerand a small etendue.

We show below that a class 3R system can be obtained with an outputpower greater than 5 mW and an etendue smaller than 0.2 mm².

We detail below how different conditions (average exposure vs pulsedexposure vs repeated pulsed exposure) drive certain designconsiderations for a scanning eye-safe laser system.

Average Exposure

At every point of the scan pattern an accessible emission (defined inthe IEC 60825-1 standard as “level of radiation determined at a positionand with aperture stops (when the AEL is given in units of Watts orJoules) or limiting apertures (when the AEL is given in units of W·m-2or J·m-2) as described in Clause 5”) averaged over the full period ofthe scan can be derived. The scanned area represents an effectiveextended object. When considering the constraints driven by an averageexposure, the maximum permissible exposure (defined in the IEC 60825-1standard as “level of laser radiation to which, under normalcircumstances, persons may be exposed without suffering adverseeffects”) may increase as the total area illuminated by a given power isincreased. Instrument optics generally limit the maximum angulardeviation that can be achieved for a scan pattern, and thus canestablish a practical or maximum achievable scan area. We cancharacterize the fill factor as the ratio of illuminated area to maximumachievable scan area. A low fill factor may not be desirable in somecases because it implies that the average laser energy is concentratedonto a few points within the maximum scan area. Since the averageexposure of the points receiving the highest average exposure may driveeye-safety considerations, patterns with higher fill factors withuniform exposure across the maximum scan areas are usually desirable.Examples of low fill factor trajectories are straight lines, circles, orellipses. Examples of high fill factor patterns are 2D rasters (withinterline spacing close to the instantaneous spot size) or Lissajouspatterns.

In addition to the fill factor of the pattern, the uniformity of theillumination can be important, for the same reason that the averageexposure of the points receiving the highest average exposure driveseye-safety considerations. In practice this means for example that araster or Lissajous patterns with triangular functions are preferable tosimilar patterns with sinusoidal functions, since they result in moreuniform intensities: for a triangular raster, the laser dwells less atthe end of each line where the scan direction is reversed, compared to asinusoidal raster where the scan speed slows progressively to zero atthe end of each line before reversing direction and slowly acceleratingback. In general, average accessible emission is evaluated at differenttime scales and over different areas in order to find the mostrestrictive conditions. In practice, the most restrictive conditions areoften be found near the edges and corners of a scan pattern where thescan speed is lowest and where two or more successive exposures canoccur in a short time frame.

A pattern's uniformity may be affected by the intrinsic properties ofthe pattern itself, but also by possible system throughput modulationsas a function of scan angle. For example, the output laser power may bereduced at the edges of a raster pattern by optical vignetting orspatial filtering. Additionally, the laser power may be directlymodulated following a pattern synchronized with the scan, to reduce thepower in some specific regions of the scan pattern. Whether specificallydesigned or not, such scan position-dependent power modulation may beconsidered when calculating the maximum permissible laser exposure for alaser system.

Pulsed Exposure

When considering a pulsed exposure, it is useful to describe an‘instantaneous’ spot size, i.e., the laser spot size that would beobtained with no scanning. If we assume a one-dimensional scan along agiven direction at a given speed (more complex scan patterns can beaddressed as a succession such short scan segments), we can consider theeffective pulse duration as the time it takes for the instantaneouslaser spot to travel across a distance equal to its diameter. The pulseduration is thus proportional to the spot size. We assume here acircular spot, but the discussion can be straightforwardly extended toirregularly shaped spots by considering its dimension in the directionof the scan. Since the angular subtense of the apparent source a is alsoproportional to the spot size and from the AEL expression in equation(2), we see that the accessible laser power (in Watts) increases withincreasing spot size according to a s^(0.75) law, where s is the laserspot size.

The duration t of the effective pulse is equal to s/v, where s is thespot diameter and v is the scan velocity on target. From the AELexpression in equation (2), the accessible laser power (in Watts)increases with scan velocity as v^(0.25).

As discussed in the case of an average exposure, non-uniformity in thescan pattern can be damaging. From the point of view of theinstantaneous exposure, a point with a slower scan velocity (assumingconstant power throughout the scan pattern) may be the most limiting.Power modulation across the scan pattern, either designed in the opticswith the use of spatial filtering or vignetting of the beam, or throughdirect modulation of the laser beam power, can be used to increase theuniformity of the exposure.

Repeated Pulsed Exposure

The previous considerations revealed that a large spot scanned rapidlyacross a wide area with close to unity fill-factor can be preferable insome cases, in the sense that it increases or maximizes the power of theRaman pump laser that can be used for a Class 3R laser system. We nowconsider the limiting case of repeated pulses: if the scan patternretraces several times through the same point, which happens at leastonce for each period of the scan pattern for a periodic pattern, anadditional correction factor should be included when calculating theAEL: C₅=N^(−0.25), where N is the number of pulses in a train of pulses.Taking the example of a standard raster pattern along a fast axis scan(e.g., 200 Hz frequency) and a slow axis scan (e.g., 20 Hz frequency), apoint along the trajectory is illuminated twice per raster frame period,i.e., twice within 1/20^(th) of a second using the examples of scan rategiven above. N is the number of pulses within the maximum time T₂ to beconsidered, with T₂=10×10^([(α-α) _(min) ^()/98.5]) seconds forα_(min)<α<100 mrad.

What this consideration implies is that, in some embodiments, thepattern can be chosen in a way that avoids retracing often onto the samespot or group of spots on the sample. As in previous considerations, thepoint that sees the most repeated exposures within a scan frame sets theupper limit on laser power. The laser power will be limited in a mannerproportional to N^(−0.25), which is a similar rule as the one derivedabove for the duration of the effective pulse in the previous case(pulsed exposure), where the laser power is limited in a mannerproportional to t^(−0.25), with t the duration of the effective pulse.

One should note that for small source (α<5 mrad) and long enough pulseduration (t>T_(i), where T_(i)=5 μs for example for wavelengths between400 and 1050 nm), C₅ should be considered equal to 1 according to theIEC 60825-1 standard. Furthermore, if α>5 mrad, one can still calculateAEL based on α<5 mrad according to the IEC 60825-1 Interpretation Sheet1: “When the class of a laser product is determined with the extendedanalysis (subclause 5.4.3) and the apparent source is larger than 5mrad, the classification may be based on a value of the angular subtenseof the apparent source less than 5 mrad (resulting in a smaller C₆ butalso larger C₅). That is, when the AE is below the AEL for an assumedsmaller apparent source, the resulting class is applicable even thoughthe image of the apparent source is larger than 5 mrad” As a result,repeated exposures may not be a concern (from a pulse exposure point ofview) for certain systems with small sources, if there a delay greaterthan T_(i) between repeated exposures. However, the average exposure maythen constraint the maximum allowable exposure for a certain laserclass.

Laser Duty Cycle

The laser may be operated in continuous wave (CW) or pulsed mode. Thelaser safety standards clearly indicate how to consider these differentconditions, and such conditions can be incorporated in the calculationsoutlined here. The laser beam scanning method described here may beeffective for both CW and pulsed laser systems.

Beam Divergence

In general, and for a fixed distance between the apparent source(defined in the IEC 60825-1 standard as: “for a given evaluationlocation of the retinal hazard, real or virtual object that forms thesmallest possible retinal image (considering the accommodation range ofthe human eye)”) and the eye, the collected energy into the eye pupildecreases as the laser beam divergence increases. However, whenconsidering Raman instruments with visible or near infrared pump lasers,the distance to be considered between the apparent source and the eyeshould be adjusted to find the most restrictive position. For example,IEC 60825-1:2014 states that “For radiation with wavelengths in theretinal hazard region 400 nm to 1400 nm, when the AEL is increased by aparameter C₆ with values greater than 1 for extended sources, it isnecessary to assess the class of the product (i.e., to compare theaccessible emission value with the corresponding AEL) at the mostrestrictive position in the beam.” In practice, the most restrictiveposition is often close to the point where the laser beam just fills theeye pupil, which may be taken, in some cases, as having a 7 mm diameter.Therefore, the reason a less divergent beam is often more restrictive onallowable laser power is generally not because of increase in thefraction of power collected in the pupil but because the image of theapparent source on the retina is smaller, concentrating the energy ontoa smaller region of the retina.

For an instrument with a fixed aperture diameter, the beam divergencedecreases with increasing standoff distance. The scan area is typicallylimited in term of the maximum deviation angle achievable by the opticalsystem. Therefore, the maximum scan area increases with increasingstandoff distance. It may desirable for practical purposes to limit thescan area so that the interrogated area overlaps well with the analyteof interest. The laser safety assessment should be realized at thestandoff distances accessible by the instrument, so that the mostconstraining condition is identified. In general, the most constrainingcondition is found at the longest standoff distance, since thesecorrespond to a lower beam divergence than closer standoff distances.Note however that the laser scanning methods presented here areespecially effective at long standoff distances.

Example of a Scanning System

FIG. 3A is a schematic of an example beam scanning device. The Ramanpump beam 300 is incident on a tip-tilt mirror 301 located in front of atelescope/telescope optics 302, formed by two lenses 303 and 304, thatfocuses the beam onto the sample 305. The Raman scattering photonsemerging from the sample 305 are collected by the same telescope 302,directed to the same tip-tilt mirror 301, which has not moved during thetime it takes for the photons to do the round trip between theinstrument and the sample (few nanoseconds). As a result, the Ramanscattering photons are reflected by the tip-tilt mirror 301 towards adetector (not shown) in a direction colinear to the initial Raman pumpbeam 300.

More generally, laser beam scanning can be achieved using the tip-tiltmirror 301 at the entrance of the telescope 302 used to focus the laserbeam 300 onto the sample 305. Assume that the telescope input is acollimated beam and that the telescope 302 has a magnification Mexpressed as the ratio between the output beam diameter to the diameterof the input collimated beam. If the entrance tip-tilt mirror 301provides a maximum angular deviation for the laser beam 300 of θ_(max),then the maximum angular deviation of the beam at the output isθ_(max)/M. This may be further reduced by vignetting in the telescope302.

Another consideration when designing a scanning system, such as thesystem/device of FIG. 3A, is to ensure that both the light from thelight source and the light received from the sample propagate along thesame path (in opposite directions) through the system, as shown in FIG.3B. Consider a mechanical frame (reference frame) in which the lightsource (Raman pump laser) and the detector (or for a Raman system, theentrance slit to a spectrometer) are fixed, for example, the mechanicalframe of the system 306 in FIG. 3B. A Raman pump laser 307 emits a beamcollimated by lens 309. That beam is fixed in the reference frame and isdirected through a dichroic beam splitter 308 toward a tip-tilt mirror301 whose orientation changes with respect to the reference frame. Thisresults in a beam of varying orientation entering the fixed telescopeoptics (303 and 304), and correspondingly the Raman pump beam is focusedto a varying point on the sample 305. There is a one-to-one relationshipbetween the tip-tilt angles of the mirror at the entrance of thetelescope and the position of the focused spot on the sample to beanalyzed.

The Raman scattering emitted from the spot illuminated by the Raman pumpbeam and its surroundings is collected by the same telescope optics 303and 304 as the one used to focus the Raman pump beam. Consequently, thatscattered light exits the telescope with the same direction as the onethe Raman beam had: the two beams are collinear. The resulting beam issent back to the tip-tilt mirror 301, which reflect the beam toward thebeam splitter 308 with the same orientation (and opposite direction oftravel) as the Raman pump beam. The Raman scattering is reflected by thedichroic mirror 308 and focused onto a slit at the input port of thespectrometer 310. An optical fiber may be used to guide light betweenthe laser and the collimating lens 309 and between the focusing lens 311and the spectrometer 310.

Upon reflection on the tip-tilt mirror 301, since the delay between thetime when the photons from the pump beam 300 impact the mirror and thetime when the scattered photons impact the mirror 301 is negligiblecompared to the time scales at which the mirror 301 moves, the mirror301 can be considered fixed. For example, for a standoff distance of 1meter, the round-trip travel time for photons is about 6 ns, while amirror oscillation period is typically in the millisecond range. Withthe mirror 301 considered fixed at the photon round trip travel timescale, the scattered photons reflect off the tip-tilt mirror in adirection collinear with the Raman pump beam. In other words, thedirection of the scattered photons reflected off the tip-tilt mirror isfixed with respect to the fixed instrument mechanical frame, includingthe spectrometer entrance slit. The scattered photons can thus beefficiently collected by a small aperture, e.g., the spectrometerentrance slit, or an optical fiber facet or other optical aperture.

Temperature Rise of the Sample

A high laser exposure on the sample is also sometimes associated withexplosion or ignition risk if the material being analyzed or a nearbymaterial is flammable or combustible. Alternatively, the sample may heatup so much under the high radiance of the Raman pump that it emits anincandescent glow that interferes with or altogether prevents themeasurement. From this point of view, there is also a desire to reducethe radiance on the sample, which is addressed by the system presentedhere.

In addition to risks of sample explosion or ignition, it may bedesirable to avoid a significant temperature rise of the sample oranalyte in order to prevent a perturbation of a chemical reactionoccurring in the analyte, for example by altering the possible reactionspathways or altering the reaction kinetics.

Local temperature heating of the analyte may also give rise toconvection flows or eddies in the analyte, potentially disturbing themeasurement by introducing a dynamic process and material variationsover time.

Fail-Safe Mechanism

The IEC 60825-1 standard states: “Laser products intended to emitscanned radiation and classified on this basis, shall not, as a resultof scan failure or of variation in either scan velocity or amplitude,permit human access to laser radiation in excess of the AEL for theassigned class, unless exposure of people is not reasonably foreseeableduring the time interval between failure and when the scanning safeguardreduces emission to levels below the AEL of the class of the product.”

The paragraph above indicates that a fail-safe mechanism shouldinterrupt the laser if a failure of the scanning mechanism is detectedand if the safety class of the system relies on the scanning systemproper operation. This can be realized by adding a secondarysystem/device monitoring the movement of the parts responsible for laserbeam scanning. For example, if beam scanning is achieved using atip-tilt mirror at the entrance of the telescope as illustrated in FIGS.3A and 3B, a laser diode can be used to illuminate the tip-tilt mirror,with the reflection directed towards a four-quadrant detector. Thesignal output from such a detector can be processed to derive theposition of the mirror, and thus ensure that the beam is being scannedas expected.

Aspects of such a scan monitoring system/device can be directed tomonitoring that:

-   -   The beam has moved across a distance equal to its diameter in a        time short enough to ensure that the maximum permissible        exposure has not been surpassed based on the pulsed exposure        calculation;    -   The beam is not re-exposing a spot more than expected, ensuring        that the maximum permissible exposure has not been surpassed        based on the repeated pulsed exposure calculation;    -   The beam is scanned following a pattern such that the maximum        average exposure at any point does not exceed the maximum        permissible exposure based on the average exposure calculation.

This implies that the scan monitor should have a fast response time,typically on the order of the time it takes for the laser spot to travelacross a distance equal to its diameter, and a memory of previouspositions at least sufficient to store one full scan period.

In addition to ensuring that limits for a certain laser class are notexceeded, a monitoring and fail-safe mechanism can be used to ensurethat the risk of sample explosion or ignition is properly mitigated.This can be achieved using the same mirror monitoring device describedabove, with a similar fail-safe control system.

FIG. 5 is a schematic of an example fail-safe mechanism that can beassociated with any Raman system/device described herein. The beamemitted by the Raman pump laser 500 is scanned using a beam scanningmechanism 501 (e.g., galvanometer scanner(s) and directed to an opticalsystem 502 (e.g., focusing optics) that focuses the beam onto a sample503. The beam scanning mechanism 501 is monitored by an independentmonitor system/device 504 that measures the angle and/or position of thebeam scanning mechanism. The signal output is analyzed by a processor505, which decides whether the movement of the beam scanning mechanism501 is within acceptable bounds to ensure safe operation (for example,for eye safety and/or explosion mitigation). If the movement is outsideof pre-programmed bounds (e.g., if an angle of a galvanometer scannerexceeds a predetermined threshold), the processor 505 can control aswitch 507 to cut the power 506 to the Raman pump laser 500.

The reaction time of the fail-safe system can be fast enough to ensurethat the accessible emission limit (AEL) (defined in the IEC 60825-1standard as: “maximum accessible emission permitted within a particularclass”) for eye-safety for a particular laser class is not reached. Anexact calculation of the accessible emission with a failed scannerenables determination of the maximum allowed reaction time, but ingeneral a reaction time equal to the time it takes for the laser spot tomove by a distance equal to its diameter (the distance is measured inthe focus plane, on the sample) may be sufficient. A slower reactiontime may be allowed as indicated in the IEC 60825-1 standard: “Forexample, a scanning safeguard may not react fast enough to preventemission above the AEL during the fault condition; however, this mightbe acceptable based on the results of a risk analysis.” (Paragraph 5.1Note 2).

Additionally, we describe below a different method to ensure that therisk of sample explosion or ignition is properly mitigated, as can becarried out by example systems generally illustrated in FIGS. 8A and 8B.It is understood that while some instances describe the target as asample while others describe the target as the pupil of an eye of asubject/person, requirements/demands on the Raman systems as disclosedherein can be applicable to tailored to any suitable target using theapproaches described herein.

FIG. 8A is a schematic of a standoff sample temperature monitoringsystem 800. The system 800 includes a Raman laser 801 that emits a beamfocused onto the target 803 using a first optical system/setup 802. Asecond optical system 804 collects an infrared thermal emission from thesample area illuminated by the laser and focuses it onto an infrareddetector 805, which may be an array for local imaging, or a single pixeldetector.

FIG. 8B is a schematic of another embodiment of a standoff sampletemperature monitoring system 806 that can be used with any Raman systemdescribed herein. The system 806 includes a Raman laser 807 that emits abeam focused onto the sample 809 using the optical system/setup 808. Thesame optical system 808 is used to collect the infrared thermal emissionfrom the sample area illuminated by the laser 807, which is thenseparated from the Raman laser light and Raman scattered photons using adichroic beam splitter 810 and focused onto an infrared detector 811.

More generally, an infrared thermometric device (e.g., the detector 805or 811) pointing at the sample can be used to ensure that the sample isnot heated beyond a certain limit above ambient temperature. The measureof the sample temperature at the point illuminated by the laser can berealized using an infrared camera or a single pixel infrared detector:the infrared thermal emission from the sample area illuminated by thelaser is focused onto an infrared detector. The intensity of themeasured signal can be related to the sample temperature with propercalibration.

The optical system for such a camera or detector can be a separatesystem, or it can share the same optics as the main Raman system, ifallowed by transmission and dispersion properties of the material used.For example, a reflective telescope may be used which can efficientlyimage the spot illuminated by the laser onto an infrared camera ordetector, as well as focus the Raman laser (for example, with awavelength of 785 nm) onto the sample and collecting the scattered Ramanphotons (for example, in the NIR band). The infrared thermal emission,the Raman laser and the scattered Raman photons can be routed throughthe beam scanning device to ensure that they all are focused from or areoriginating from the same point on the sample.

The sensitivity, accuracy, and time response of the camera or detectorshould be selected to ensure adequate measure of a potential temperaturerise and allow for the system reaction is a certain limit is exceeded. Atypical off-the-shelf infrared camera can measure temperaturedifferentials in the tens of milli-Kelvin range, whereas it is expectedthat an accuracy of 0.1-1 K should be sufficient to monitor a potentialtemperature rise of the sample.

FIG. 9 is a schematic of another example fail-safe mechanism (e.g., forexplosion or ignition risk mitigation) based on measurement of thesample temperature and can be used with any Raman system describedherein. The beam emitted by the Raman pump laser 900 is scanned using abeam scanning mechanism 901 and directed to an optical system 902 thatfocuses the beam onto a sample 903. The temperature of the illuminatedarea of the sample 903 is measured, either in absolute terms or relativeto the surrounding non-illuminated material, using a calibrated infraredthermometric device (not shown). The signal output is analyzed by aprocessor 905, which decides whether the sample temperature is under acertain threshold or not. The threshold may be an absolute temperature,or a relative temperature compared to ambient or compared to thesurrounding sample material. If the temperature is above a certainthreshold, a switch 907 cuts the power 906 to the Raman pump laser 900,preventing emission of more light from the Raman pump laser 900. Thereaction time of the fail-safe system should to be fast enough to ensurethat the maximum allowed sample temperature (absolute or relative) isnot reached.

Two different thresholds may be used to trigger the automatic shut-offof the laser 900: (1) a relative temperature rise, compared to ambientor compared to neighboring, non-illuminated material, or (2) theabsolute temperature of the sample, which may be known to ignite orexplode above a certain temperature. These thresholds may be informed byexperiments, and may be dependent on the sample being measured, or theenvironmental conditions surrounding the sample (temperature, pressure,presence of oxygen, humidity, presence of flammable materials, etc.)

In another temperature measurement method, any suitable Ramanspectroscopy system described can be used to determine the temperatureof the analyte, either by observing a shift of a narrow Raman line, orby measuring the ratio of the signal strength for a Raman band, observedat the Stokes and anti-Stokes positions. The second measurement methodrelies on the observation of both a Stokes and an anti-Stokes signal fora given Raman band. This is usually easier for a band with a low Ramanshift, since the signal strength of the anti-Stokes peaks decreasesexponentially with increasing Raman shift.

FIG. 10 generally illustrates this approach and shows the differentRaman signals that can be observed. Typically, the Stokes Ramanscattering 1014 is used for chemical analysis since the anti-Stokespeaks (see anti-Stokes Raman scattering 1010) are much weaker. If aRaman peak with a low Stokes shift is present, and if allowed by thebandwidth of the Raman filter used to suppress the Rayleigh scattering1012, the corresponding anti-Stokes peak may be observed. The ratio ofamplitudes between the two peaks (anti-Stokes and Stokes) may then beused to measure the temperature of the sample.

A Raman temperature marker may be added to the analyte to facilitate thetemperature measurement using such approaches. The temperature markermay be a material with one or several of the following features: (1) amolecule (e.g., a diatomic molecule, such as O₂) having a simple Ramanspectrum, (2) a molecule having at least one strong Raman peak with alow frequency shift (<500 cm⁻¹) (e.g., a diatomic molecule with heavyatoms or a material such as sulfur), (3) a chemically inert molecule,(4) a material that can be easily separated from the analyte afteranalysis (e.g., magnetic nanoparticles), and/or (5) a material with asimple Raman spectrum (one or few isolated Raman peaks) that is easilyseparable from the spectrum of the analyte of interest (e.g. Silicon(Si) particles). Such a Raman marker can be added to the analyte beforeanalysis. By providing a strong Raman band with a low Raman shift, itcan allow for temperature measurement based on the ratio of the Stokesand anti-Stokes signals for this band. The marker may then be extractedusing standard chemistry separation techniques. A chemically inertmarker is desirable so that it does not interfere with the reaction, forexample, in the case of reaction monitoring in a reaction vessel.

FIG. 11 illustrates the use of a Raman temperature marker fortemperature measurement of the analyte. (1) Consider a reaction vesselcontaining one or a few analytes. At (2), a Raman marker material, whichcan be any suitable material as described above, is added. This materialhas specific characteristics for measuring the temperature of the slurryor liquid in the vessel. At (3) the measurement is realized, which canpossibly include, but is not limited to, continuously monitoring areaction. The Raman signal from the Raman marker allows measurement ofthe temperature of the analyte. (4) The Raman marker is separated fromthe rest of the analyte after analysis.

Embodiment of a Fail-Safe Mechanism

We consider a laser system with a beam scanning optical system that ismonitored so that the position of the scanning optics (e.g. tip-tiltmirror) is recorded over time, and the data is available for quasi-realtime processing and analysis. FIG. 4A is a schematic of a mirrormonitoring device and illustrates a light source 400 (e.g., a lightemitting diode or laser) that emits a beam 401 directed at the tip-tiltmirror 402 in a direction that is typically different from the directionof incidence of the Raman pump beam to avoid interfering with thetransmission of the Raman pump beam. The reflected light is directed toa four-quadrant detector 403.

FIG. 4B illustrates time signals 404 (upper plot) and 405 (lower plot)from the four-quadrant detector (with quadrants A, B, C, D) that allow aprocessor (not shown) to evaluate the angle of the tip-tilt mirror 402in real-time, and consequently to evaluate whether the beam scanning isoccurring in a manner consistent with the expected pattern. Thefour-quadrant detector provides a signal proportional to the lightintensity impinging on each quadrant (A, B, C and D) and a processorcomputes the three following signals: the sum signal from all fourquadrants as a measure of total beam intensity, the difference betweenthe top and bottom halves (A+B−C−D) indicating position of the beamalong the top-down axis, and the difference between the left and righthalves (A+C−B−D) indicating the position of the beam along theleft-right axis.

Thus, generally, a secondary laser (e.g., the light source 400) incidenton the optics with its deflection measured with a four-quadrant detector(the detector 403) is one embodiment of such monitoring system. Thesystem may keep in its memory the history of the position(s) reached bythe beam scanning optics, which can be mapped one-to-one to thepositions reached by the laser spot on target. If the power of the laseris known, potentially as a function of deflection angle of the beamscanning optics, a map of total energy deposited at each point withinthe instrument scanning range or field-of-view can be calculated. Thismap can be updated by incrementing the corresponding total energy forthe spots successively exposed as the beam is scanned. At each spot, theenergy deposited can be compared to the maximum permissible exposurelevel for a laser system of a certain class. If the energy depositedapproaches the maximum permissible value, the instrument can beautomatically turned off by the system embedded electronics andsoftware. Since different conditions needs to be considered (pulsedexposure, average exposure), several maps can be acquired: a mapcontaining information on instantaneous exposure, for which the energydeposited is incremented when a spot is exposed, and decremented if thespot has not been visited for more than T_(i) (defined in the IEC60825-1 standard as the time below which pulse groups are summed); and amap containing information on average exposure, acquired for exampleover a scan period. The exposure values in these maps can be comparedwith their respective maximum permissible exposures.

Potentially Limiting Cases

The beam divergence (which can be expressed as a function of the beamdiameter at the system output and of the system focal length) as well asthe laser average power and wavelength are also used to determine eyeand/or sample safety. We consider here two limiting cases fordetermining the remaining system parameters:

-   -   In the limit of a fast laser beam scanning (as fast as        possible), the ‘pulsed exposure’ AEL becomes higher than the        ‘average’ exposure AEL. In other words, the average exposure may        limit the laser power of the system for a certain class.        Affected design parameters include the total span and fill        factor of the scan pattern (i.e., the total illuminated area, if        we assume a uniform exposure).    -   In the limit of a slow laser beam scanning (as slow as        possible), the pulsed exposure may limit the laser power. In        that case, affected parameters include the laser spot size and        the scan speed.

Scanning to Reduce Risk of Ocular Damage or Explosion

An example embodiment of such a Raman system is shown in FIG. 1, whichillustrates a scanning standoff Raman system 100 being used on a targetsample 103. The system 100 generates and scans the Raman laser beam 101over the target sample 103 with a predetermined pattern 102 and speed tomitigate laser eye-safety (ocular damage) or risk of explosion due totemperature rise of the sample 103. If the scan pattern has a high areafill-factor with high linear scan speed, the effective laser spot maybecome indistinguishable from that produced by spatially incoherentsources, such as light emitting diodes or flashlights. The increase insurface temperature of the sample 103 caused by laser beam illuminationwould be lower with such a scanned pattern 102 than without scanning;hence mitigation of explosion of a target sample due to temperaturerise. Similarly, the image of such a scan would produce a lowertemperature increase at any point on the retina of an observer comparedto the temperature increase obtained without scan; hence the increase ineye-safe laser power.

FIGS. 2A-2C indicates some examples of scan patterns. A low fill-factorscan pattern shown in FIG. 2A would heat selected portions of thesurface of the sample or the retina of the eye more than the higherfill-factor patterns shown in FIGS. 2B and 2C.

The patterns with higher fill-factor (ratio of illuminated area to totalarea encompassed by the pattern) are more effective at reducing heatingdue to absorption of laser power by the sample or retina. Specifically,the patterns of FIGS. 2B and 2C have higher fill factors than thepattern of FIG. 2A and spread the thermal load over a larger area,reducing the likelihood of thermal damage to an observer's retina or therisk of explosion of the sample. As illustrated in FIG. 2C, the patterncan be a Lissajous pattern with a=5 and b=4, where a and b are used todescribe the pattern as a graph of a system of the following parametricequations: x=A sin(at +δ), y=B sin(bt).

The example patterns of FIGS. 2A-2C aside, any suitable scan patternpermitted by the scanning system/optics can be employed. For a givenslow axis frequency (b for example), the fast axis frequency (a) may bechosen to ensure enough fill of the pattern, considering the spot sizeand the total scanned area. For example, a=10b or a=25b may be practicaland desirable. Having a be an integer multiple of b can be advantageousto ensure that the scan pattern is periodic with frequency b. Suchrepeatability can be useful to synchronize the processes used to acquirea Raman spectrum.

The dwell time of the spot at each point can be chosen to be smallenough to satisfy the eye safety requirement of a given laser class.Faster linear scan for any pattern reduces thermal damage at any givenspot on the retina or risk of sample explosion.

In some cases, scan patterns where scan lines take more time to comeclose to each other would reduce thermal crowding and may be preferred.For example, in the Lissajous pattern of FIG. 2C, the folding lines arefather away for longer time before coming close to each other comparedto the raster pattern in FIG. 2B, where each row is followed by anadjacent row.

Consider the following system specifications: An instrument with a 12-mmdiameter beam at the system output, focusing to a spot on the samplelocated 80 mm in front of the instrument. The tip-tilt mirror used toscan the beam has an oscillation period of 100 Hz along the fast axis,and a mechanical maximum deviation angle of 4 degrees. The correspondingilluminated area on the sample, averaged over a mirror scan period, isabout 0.9 mm×0.9 mm. The system is configured to have an instantaneousspot size of 0.15 mm. The laser peak power is 100 mW (66 mW averagepower), and the wavelength is 785 nm. The system etendue as definedabove is G=3×10′ mm². We consider an evaluation condition such that theeye is located 10 cm directly in front of the focused spot. Under theseconditions, we find that the system is at ˜73% of the accessibleemission limit (AEL) for a class 3R pulsed and repeated pulsed source(which is how we model the instantaneous exposure), and at ˜87% of theAEL for a class 3R system when the average effective extended source isconsidered. Contrast this to the case when the scan motion is stopped:in this case, the system's AEL is five times higher than the AEL for aclass 3R system.

Accordingly, any Raman system described herein can be outfitted with amonitoring system/component that is coupled to beam steering elementssuch as the tip-tilt mirror and/or to the laser, and can detect amalfunction that would cause the laser beam, when having a first lasersafety class, to exceed a maximum permissible exposure for a secondlaser safety class which is lower than the first laser safety class. Thesecond safety class can be, for example Class 3B or 3R according to theIEC 60825-1 standard, or class 1, etc. The result can be that thismonitoring system can attenuate (e.g., via a controllable filter),redirect (e.g., via a controllable mirror), block, and/or turn off thelaser beam in response to this detection. In some cases, as detailedherein for FIGS. 8A and 8B, the monitoring system/component can takesuch actions upon detecting that the temperature of the sample is abovea threshold value.

Handheld Standoff Differential Raman Spectroscopy

Drawbacks of Conventional Handheld Raman Spectroscopy Systems

As noted above, Raman spectroscopy has proven to be a highly valuableportable analytical tool for accurate chemical identifications. The mostcommon implementation, the operator must hold the Raman spectrometerclose (less than couple of centimeters away) to the substance underinvestigation, illuminating the substance with a pump laser andcollecting the Raman scattering.

Existing handheld Raman systems that aim at providing a fieldablecapability for rapid chemical analysis of solids and liquids typicallyhave a very short standoff distance—less than 1 inch in most cases.There exist a few reasons for the use of such small measurementdistance:

-   -   In part, this is to maintain a small instrument form factor by        using a small focusing lens at the instrument output: however, a        smaller lens provides a smaller collection efficiency for the        scattered Raman photons if the standoff distance was increased.    -   Since the systems are typically classified as Class 3B lasers        for eye safety, having a small measurement distance allows the        use of a shield that screens potentially harmful radiation        reflecting from the sample being analyzed, and prevents such        radiation from reaching the operator's eyes.    -   In many cases, the systems are designed to contact the samples,        conveniently maintaining the distance between the instrument and        the sample to ensure optimal focusing and correspondingly        increasing or maximizing the signal-to-noise ratio.    -   The small standoff distance makes it convenient to shield the        sample from ambient light, which can create a background signal        that can interfere with the Raman signal.

The drawbacks of such a small standoff distance include:

-   -   The operator needs to spend time setting up the measurement,        carefully positioning the instrument in close proximity to the        sample such that the focused Raman probe is on the sample (or in        the sample in the case of a liquid), which sometimes requires        moving obstacles or clearing space around the sample to properly        position the instrument. In addition to requiring time, this        task can also be dangerous as it presents risks of spilling        chemical containers or triggering chemical reactions in the        process of clearing space around a sample in a cluttered scene.        An instrument with an increased standoff distance would        facilitate measurements in such crowded environments.    -   Since the operator needs to bring the instrument close to the        sample, the operator herself usually needs to be close to the        sample, which may be a toxic compound or present an explosion        risk. The measurement itself may trigger an explosion of the        sample, further endangering the operator. This risk is sometimes        mitigated with the use of a time-delayed measurement, which        further increases the overall measurement time. By increasing        the distance between the operator and the sample, an instrument        with an increased standoff distance would increase operator        safety.    -   If the instrument is close to the sample, the instrument (and        the user) may become contaminated by the sample or surrounding        chemicals. An increased standoff distance may decrease the risk        of contamination of both the user and the instrument. And with        an increased standoff distance, the sample (or the instrument)        can be placed in an enclosure to further reduce the risk of such        contamination. For example, the sample may be left in a fume        hood or a closed sample compartment, while the instrument can        measure through the closed window of a fume hood or the        (transparent) walls of the sample compartment.    -   In some cases, the instrument cannot be brought close to the        sample, e.g., the sample may be at the bottom of a container        with a narrow opening, behind a glass or plastic barrier,        contained in a thick-walled container, etc. Instead of opening        the corresponding containers, windows, fume-hood sash or other        obstacles, an instrument with an increased standoff distance        would provide a way to realize the measurement rapidly and        directly through the obstacle.

In addition, most existing handheld Raman analyzers have difficultyanalyzing samples with strong fluorescence (or photoluminescence). Forthese samples the Raman signatures are often much smaller than a large,broad fluorescence background that carries limited chemical-specificinformation. That background is largely un-helpful in identifying thechemicals measured, and the shot noise associated with that backgroundcan overwhelm the Raman signatures. The background may also be modulatedby the instrument transfer function, producing small ripples in thesignal that can be difficult to separate from the Raman signal usingbackground suppression methods.

A typical solution to the problems caused by this background is the useof a longer wavelength for the Raman pump laser since the fluorescenceof many materials scales down with increasing wavelength. For example,systems with a pump wavelength of 1064 nm have been developed to haveimproved performance in the presence of strong fluorescence compared tothe more common instruments using a 785 nm pump wavelength. However, theuse of a longer wavelength can come at the expense of a poorer detectorperformance. Instruments with a pump wavelength 1064 nm typically useIndium Gallium Arsenide (InGaAs) detectors with much higher dark noisethan the silicon detectors used in 785 nm pump-wavelength instruments.

Raman microscopes are commonly used in laboratories to analyzespectroscopically small samples (e.g., single or few grains orparticles, fibers, hairs, etc.) or to map the chemical composition of aheterogenous solid mixture (e.g., a pharmaceutical pill). Theseinstruments are typically table-top units and use typical microscopeobjectives with short working distances. To ensure eye-safety, theinstruments are often enclosed in an enclosure opaque to the laserwavelength used as the Raman pump. Alternatively, the sample compartment(instead of the full instrument) may have a movable or articulated panelthat allows the compartment to be closed to prevent light from the Ramanlaser from escaping the sample compartment and the rest of theinstrument. These enclosures often also serve as a shield for ambientlight, preventing the room lights from reaching the sample and frombeing collected by the instrument optical system. Reducing thisbackground signal facilitates the spectral analysis of the Ramansignatures. Such a microscope often uses an eye-piece or a digitalcamera to view the sample and align the Raman pump laser to specificfeatures of interest, such as individual particles. To obtain an image,the sample can be moved under a fixed Raman laser spot, using amotorized two-axis (x-y) stage or the Raman spot may be scanned over thesample.

These instruments suffer from a few limitations, including:

-   -   It is sometimes undesirable to confine the sample, or all or        part of the instrument, in an enclosure to provide eye-safety        and eliminate ambient light. For example, it may be advantageous        for the operator to be able to manipulate the sample, hold a        part of the sample, or re-position the sample, while rapidly        triggering measurements on demand, e.g., when a portion of the        sample of interest is at the location of the Raman laser focus.    -   The measurement of very fluorescent samples is often difficult        because of the absence of fluorescence mitigation.    -   The measurement of dark, colored, or very absorptive materials        can be difficult because of problems linked to the local heating        of the sample due to absorption of the Raman laser energy.        Potential issues include ignition or explosion of the sample, or        incandescent glow from the sample saturating the detector.

A Raman spectrometer can also be used as a chemical analysis tool tomonitor chemical synthesis. For such an instrument, an increasedstandoff capability compared to existing instruments can allow todirectly probe the reagents inside a reaction vessel, whether directlythrough the walls of the vessels in the case of transparent plastic orglass vessels, or through observation ports with a glass or plasticwindow, or through other openings in the vessel. Whereas a Raman probeis currently often inserted in the vessel and into the reagents, whichcan cause contamination issues and may not be practical if severalvessels or reactions are to be monitored with one instrument, a standoffinstrument does not use a probe inserted into the vessel or chamber orinto the reagents themselves. This is attractive because it reduces oreliminates contamination. Such instruments could also benefit fromfluorescence mitigation since some reagents may have strongfluorescence. Additionally, shielding the light at the point ofmeasurement may not always be practical in a well-lit chemistrylaboratory. Doing so could create unpractical obstructions around thereaction vessel, possibly adding safety hazards. Finally, a solution forincreased eye safety and explosion mitigation is of interest since somereagents or synthesis products may be explosives or flammables, havingthe potential to be ignited by the Raman laser.

Handheld Raman Spectrometers

FIG. 14 is a block diagram of an example handheld differential Ramanspectroscopy system 1400. The system includes a Raman pump laser 1402that couples to a probe 1404 external to a sample 1406. A telescopesystem 1408 is employed to couple the pump beam from the laser 1402 tothe sample 1406, and to receive emission from the sample 1406. A camera1408 coupled to the telescope 1408 can be employed for various purposesas described herein including, but not limited to, image stabilization,sample temperature monitoring, etc. The system 1400 also includes aRaman spectrometer 1412 with a dispersive element, such as a grating orprism, and a camera (e.g. a CCD or CMOS array) 1414 for receiving theRaman signal from the sample 1406 and for generating the Raman spectrumtherefrom.

The system 1400 also includes a controller/processor/central processingunit (CPU) 1418 for receiving the digitized Raman spectrum from thedigitizer 1416, and for presenting a representation of the spectrum on auser interface 1420, such as a display screen of the user interface. Asillustrated in FIG. 14, the user interface 1420 can encompass multipleinterface elements including, but not limited to, a display screen,touch screen, one or more switches, indicator lights, and one or severalvisible pointing lasers used to give user feedback on the aim and/orfocus of the system.

The system 1400 can also include various components for control and/oroperation of the components noted above. For example, as illustrated inFIG. 14, the CPU 1418 can control operation of the laser 1402 using alaser driver 1422. A thermo-electric cooler (TEC) 1424 a, and itscorresponding TEC control 1226 a, are coupled to the laser 1402 andcontrollable by the CPU 1418 keep the temperature of the laser 1402within predetermined ranges. Similarly, a TEC 1424 b and a correspondingTEC controller 1426 b are coupled to the CCD 1414 and controllable bythe CPU 1418 to control the temperature of the CCD. A motor controller1428 is coupled to the CPU 1418 to permit control of a motor 1430 thatmanipulates one or more optical components associated with the telescope1408 such as, for example, a tip-tilt mirror as shown in FIGS. 3A and3B.

FIG. 14 also illustrates a power supply 1432 for powering components ofthe system 1400, and an input/output interface 1434 for communicationwith other devices/systems and including, for example, one or more USBports, a GPS receiver, etc.

More generally, an example handheld, standoff Raman spectrometer caninclude (1) a Raman pump laser, (2) an optical system/telescope focusingthe laser output onto the sample and collecting the scattered Ramanphotons, (3) a spectrometer analyzing the collected scattered light andoutputting the Raman spectrum, (4) electronics and power sub-systemssupporting operation of the above components. The optical system mayinclude a beam scanner to scan the beam for eye safety andexplosion/combustion mitigation as described above. The spectrometertypically includes an entrance slit, a first lens collimating the lightemerging from the slit and directing the light to a fixed diffractiongrating, a second lens to focus the light diffracted by the grating ontoan image sensor. In typical operation, the laser source (Raman pumplaser) is turned on and the CCD integrates the signal received in thedifferent pixels, each of which corresponds to a different wavelengthbin. At the end of a set integration time, the electronics read theimage sensor to obtain a Raman spectrum. The operation can then berepeated to average the measurement over N frames. Finally, the laser isturned off. We note that the image sensor may be a CCD camera, a CMOScamera or other similar image acquisition hardware. In the rest of thedescription, the use of CCD to describe the image sensor is not meant tobe prescriptive of a particular type, and alternative image sensors maybe used.

FIGS. 12A-12E are a collection of schematics representing differentmeasurement conditions for a standoff Raman analyzer, or otherlaser-based, standoff spectrometers. FIG. 12A illustrates standoffmeasurement of a solid sample at its surface. FIG. 12B illustratesstandoff measurement of a liquid in a container through the containerwall. The container should be at least partially transparent to theRaman wavelength and the neighboring Stokes range. In addition, oralternatively, FIG. 12C illustrates standoff measurement of the liquidin the container through the container opening. FIG. 12D illustratesstandoff measurement of an analyte in a reactor such as, for example,through a port (e.g., window) in the reactor wall. The instrument may bepositioned in direct contact with the reactor wall or spaced therefrom.FIG. 12E illustrates measurement of an analyte in a flow cell.

The system is designed to allow for handheld operation. The designaccommodates drifts in the operator's aim during the measurement. Thisdrift changes the location of the interrogated spot on the sample overthe duration of the measurement, leading to potential fluctuations inthe measured Raman signal.

FIG. 13A is a photograph of an example handheld Raman standoffdifferential spectroscopy system with an internal beam scanner thatreduces the risks of ocular damage and sample explosion or combustion.FIG. 13B illustrates the handheld Raman standoff differentialspectroscopy system during use, when measuring a white powder throughthe closed window of an oven.

Dynamic Background Subtraction

The integration/frame time is short (<0.5 s), and a background frame isinterleaved between successive Raman frames. Here, “background frame”refers here to a CCD frame for which no Raman laser illuminates thesample during the frame integration time. In contrast, a Raman frame isobtained when a laser (the Raman pump laser) illuminates the sampleduring the frame integration time. The fast interleaving of backgroundframes allows the electronics to subtract the effects of ambient light,even when the background due to ambient light fluctuates, because ofmodulation of the ambient light source itself or because of theoperator's hand movements.

Difference Raman Spectroscopy

FIG. 15 illustrates an example difference Raman spectroscopy system 1500with two or more lasers. It is understood that, unless explicitly notedotherwise, components of FIG. 15 may be functionally and/or structurallysimilar to similarly named components of FIGS. 14, 16. For example, thetelescope 1408 may be similar to the telescope 1508, and so on. FIG. 15illustrates a bank of lasers 1502 a, 1502 b . . . 1502 n (with lasers1502 a, 1502 b shown for simplicity), and an optical switch 1530 forswitching between lasers.

Using the scenario where the system 1500 includes two lasers 1502 a,1502 b, the system 1500 described here uses two Raman pump laser beamswith closely spaced wavelengths: the system is a differential Ramansystem, related to what is sometimes referred to as Shifted ExcitationRaman Difference Spectroscopy (SERDS). A frame is acquired with thefirst Raman laser beam (e.g., the laser 1502 a), then a second frame isacquired with the second Raman laser beam (e.g., the laser 1502 b). FromKasha's rule, the fluorescence signal is similar between the two framesin the limit of a small difference in excitation wavelength.Consequently, subtracting two frames eliminates the fluorescence signal,but preserves the Raman information because the Raman spectrum shiftswith a shift of the excitation wavelength. In some cases, thefluorescent background is not entirely removed, if for example the powerof the two Raman lasers is not exactly the same, or if there is a slightdifference in absorption between the two Raman lasers. Even if thefluorescent background is not entirely cancelled, the reduction of itsamplitude with respect to the Raman signature will greatly assistanalysis of the Raman signal.

In other words, the difference Raman technique involves acquiringsuccessively the Raman spectra of a sample using two pump lasers withclosely spaced wavelengths or a single laser that emits two pump laserbeams with closely spaced wavelengths in quick succession. The two laserbeams excite a similar fluorescence response from the sample, but theRaman signatures are shifted, allowing one to extract the chemicallyspecific ro-vibrational (Raman) information from non-specific chemicalfluorescence. The technique is compatible with the interleaving ofbackground spectra measurements to efficiently subtract ambient lightfeatures (e.g., room lights, sun), as described above. In this case, thedifference spectrum may be obtained as the difference of twointermediate spectra, one obtained as the difference of a first Ramanspectrum and a background spectrum (which can be acquired when, forexample, neither laser 1502 a not laser 1502 b is operating), the secondobtained as the difference of a second Raman spectrum and a backgroundspectrum. The background spectra may be identical for both sets. Sinceeach intermediate may be processed, which includes for example removalof cosmic ray signatures, as well as scaling of the spectra along theintensity axis, or shifting of the spectra along the wavenumber axis,the information from the background spectrum does not necessarily cancelout when the final difference spectrum is computed. In general terms, adifference spectrum can be calculated from the information contained inthe three spectra potentially acquired (first Raman spectrum, secondRaman spectrum and background spectrum).

Difference Raman spectroscopy can be used to remove any commonbackground between spectra acquired with different Raman pumpwavelength. This includes fluorescence of the analyte or of thesubstrate, fluorescence from internal instrument optics, ambient light,blackbody emission from a hot analyte or substrate, and dark currentfrom different CCD pixels.

More than two laser beams can be used for difference Raman spectroscopy,as illustrated in FIG. 15. As long as the wavelengths of the differentlaser beams used are close enough, such that Kasha's rule remain validand the fluorescent background is common to the spectra obtained withthe pump laser beams, the component of the spectra due to Ramanscattering can be extracted from the common background (fluorescent,photoluminescence, or ambient light). In some cases, the differencebetween wavelengths of successive lasers can be about 0.25 nm, about 1nm, about 2 nm, about 5 nm, about 10 nm, including all values and subranges in between.

The Raman laser beams used for difference Raman spectroscopy may beemitted by two or more individual lasers, either separately packaged orco-packaged into a single sub-system, or by a single tunable laser thatis successively tuned between two emission wavelengths, using a thermalcontrol (e.g., modulation of the laser heat sink temperature),electrical tuning (e.g., modulation of the laser drive current),mechanical tuning (e.g., rotation of a frequency-selective feedbackgrating), etc. The lasers may be semiconductor laser diodes withexternal feedback gratings for frequency stabilization. The laser(s) maybe Distributed Bragg Reflector (DBR) laser(s) or Distributed Feedback(DFB) laser(s) whose output wavelengths can be tuned by varying theinjected current (the refractive index of the semiconductor material maybe affected by the current injected in the DBR) or the heat sinktemperature.

When multiple lasers are used, it may be desirable to combine theiroutputs so that the beams illuminate the same spot on the sample. Sincethe lasers illuminate the sample sequentially, this beam combining canbe achieved using a switch, such as the optical switch 1534. Forexample, an opto-mechanical switch can be used to select which laser'soutput is directed to the input port of an optical system that focuseslight onto the sample. Such switch can be a fiber-coupled switch, havingtwo fiber-coupled lasers as inputs and a single fiber as an output,directing the light towards the system sampling port or probe. A thirdinput, kept dark, may be used to obtain a condition where no laser lightis directed at the sample, to measure background ambient light collectedby the system, for subsequent subtraction from the Raman spectra. Ifsuch a switch is used, the lasers may be operated in continuous wave.

Alternatively, the outputs from the different lasers used as Raman pumplasers, such as the lasers in FIG. 15, may be beam combined in apermanent, static manner. Different laser beam combining approaches maybe used: spectral beam combining, aperture multiplexing, knife-edgecombining, etc. The different lasers are permanently coupled, either infree-space or via an output fiber, such that their beams form a singleoutput beam.

To sequentially illuminate the sample with the different lasers, one maymodulate the lasers directly. In other words, the lasers may be turnedon and off by controlling their electrical power supply, allowing tocontrol if one, several or no laser(s) illuminate the sample at anygiven time. This approach can offer greater switching speed (switchingat microsecond time scale, compared to a millisecond time scale for atypical opto-mechanical switch). It also allows for more complexillumination patterns (more than one or no laser at a time) and can savepower since the lasers are consuming the most power when they illuminatethe sample.

The lasers may be kept at a constant temperature, for example, using athermo-electric cooler and its associated control electronics. Thattemperature may be the same or different between the different Ramanlasers used. The lasers may also be left uncooled (i.e., no temperatureregulation). In this case, the wavelengths of the Raman lasers may driftover time as the instrument temperature varies. The relative temperaturedrift between the different Raman lasers may be reduced or minimized bythermally connecting the lasers, either through a common sub-mount or acommon heat-sink or a thermal connection between their respectiveheat-sinks.

If the wavelengths of the Raman lasers can drift, knowledge or ameasurement of the Rayleigh scattering (zero-Stokes shift peak) in thespectrometer can be used as a reference for calculating the Stokes shiftof the Raman spectrum. In other words, the spectrometer measures thespectrum of the incoming Raman scattering in absolute units (i.e.,wavelength of the Raman scattering photons). However, the informationused to identify a chemical based on its Raman spectrum is the Stokesshift of the Raman features, i.e., the energy difference (usuallyexpressed in cm⁻¹) between the Raman scattering photons and the Ramanpump photons. If the Rayleigh scattering (also called the zero Stokesshift) is visible, that difference can be computed, even when thewavelength of the Raman pump(s) varies over time. An intermediate optionbetween constant temperature regulation and no temperature regulation isto allow the laser temperature to float within a certain temperatureranges (e.g., 30-60° C.), and to maintain the laser temperature withinthat range using a heater or cooling element.

Alternatively, other options can be used for frequency calibration ofthe Raman shift, including the use of a reference arm in the instrument,with a known chemical sample along the optical path, generating a Ramanspectrum that is known and can be used for calibration purposes.

Accordingly, in some embodiments, a method of performing Ramanspectroscopy, such as with the Raman system 1500, can include projectinga first Raman pump beam (e.g., from laser 1502 a) at a first wavelengthonto a sample 1506 (directly or through interleaving materials) from astandoff distance of at least about 1 cm, about 2 cm, about 5 cm, about10 cm, about 30 cm, about 90 cm, about 2 m, about 4 m, including allvalues and sub ranges in between. As described herein with respect toFIG. 2, the first Raman pump beam can be scanned across the sample 1506to reduce local heating. The first Raman beam can have a pulse durationof about 3 ms to about 2000 ms, including all values and sub ranges inbetween. The first Raman pump beam can irradiate the sample 1506 with atleast about 10 mW, about 50 mW, about 200 mW, at least about 500 mW ofpower, including all values and sub ranges in between.

The method also includes detecting a first Raman signal emitted by thesample in response to the first Raman pump beam. This first Raman signalcan represent a first Raman spectra/signature of the sample, and canalso represent a first background spectra/signature (e.g., fluorescencefrom the sample, ambient light, etc.). In some cases, the first Ramansignal can include the fluorescence and/or the ambient light.

The method can further include projecting a second Raman pump beam(e.g., from laser 1502 b) at a second wavelength different than thefirst wavelength onto the sample from the same standoff distance. Themethod can also include detecting a second Raman signal emitted by thesample in response to the second Raman pump beam, where the second Ramansignal represents a second Raman spectra/signature and a secondbackground spectra/signature. As described in greater detail withrespect to FIG. 17 below, the first and second Raman pump beams can beinterleaved in some cases.

The method can further include generating a post-processed signaturebased on the first Raman signal, or the second Raman signal, or both.For example, the post-processed signature can be the difference betweenthe first Raman spectra and the second Raman spectra.

The post-processed spectra/signature can have a background lower than orequal to the first background spectra/signature since, as describedabove, with both the background spectra being similar, their differencewill be small. Accordingly, other than the Raman signature, thefluorescent and/or ambient light background can also be estimated basedon the first and second Raman spectra. In some cases, the ambient lightcan be measured separately, and the post-processed spectra/signature canaccount for the measured ambient light. The post-processedspectra/signature can be used to identify the sample 1506 such as, forexample, by comparing against a library of Raman spectra/signatures ofdifferent materials. The sample 1506 may be identified as (orcontaining) a hazardous material, a combustible material that is notignited by virtue of the parameters of the first and/or second Ramanbeams, a light absorbing material that absorbs light from the firstand/or second Raman beams without overheating (i.e., beyond athreshold), etc. The sample 1506 may be within a reaction vessel asillustrated in FIGS. 23-24, and the post-processed signature canidentify at least one chemical species within.

In some cases, the system 1500 and this method can include aspects ofthe setup described for FIG. 19, and encompass illumination of thesample 1506 with at least one visible beam at the same position as theRaman beam. As described for FIG. 19, in some cases, two visible beamscan be used, with each forming a different spot on the sample. Thismethod can then include moving either the system 1500, or adjusting aworking distance thereof, to reduce the distance between the spots asdescribed for FIG. 19.

Instrument Timing and Synchronization

The difference Raman spectroscopy technique relies on the backgroundsignal (from sample fluorescence, ambient light, etc.) being commonbetween the spectra acquired successively with different pump lasers.This may be difficult in a handheld situation where the aim of the usermay be drifting over time. By illuminating different points on thesample and having varying distance between the sample surface and theinstrument focal point, the Raman signal, the sample fluorescence, thecollection efficiency of ambient light into the instrument may eachvary. Different points on the sample may also have different chemicalcompositions (e.g., if the sample is a heterogeneous mixture) ordifferent reflectivities (e.g., different colors), further contributingto the variation of the measured signals. The material under study mayalso exhibit bleaching of the fluorescence under illumination by theRaman laser, leading to a reduction of the fluorescence intensity overtime: this is another source of time variation of the fluorescencesignal.

To account for these variations, the difference Raman technique can beimplemented in a manner consistent with dynamic signals. We achieve thisby maintaining a short frame time (e.g., <0.5 seconds) for theacquisition of each difference Raman spectrum as well as the backgroundspectrum. This short acquisition time reduces or minimizes differencesbetween successive frames due to hand movement, sample variations,bleaching, etc.

The spectrometer may interleave a background frame between pairs ofdifference Raman frames. An example of acquisition sequence is asfollow: one frame is acquired with Raman laser 1 on (Raman laser 2 off),a second frame is acquired with Raman laser 2 on (Raman laser 1 off),and a third frame is acquired with no Raman laser on. We maintain ashort frame time (e.g., <0.5 s) for each frame to mitigate potentialvariations due to ambient light modulation, operator hand movements,fluorescence bleaching, and other causes of signal variations. Theduration of the frames is usually the same for all frames but may bevaried depending on exposure conditions: in the presence of highintensity ambient light (e.g., sunlight) or large sample fluorescence,it may be desirable to reduce the frame time (while keeping them allequal) to avoid saturation of the detector. The choice of frame time isa trade-off between reducing read-out noise (short frames are read outmore often for a given total integration time and therefore tend to havemore CCD read-out noise) and reducing potential variations of thesignals (generally, the shorter the frames, the more robust themeasurement is against time variation of the signal), while ensuringthat the CCD does not saturate (which may involve limiting the maximumintegration time).

The difference Raman system is compatible with an optional beam scanningof the laser source on the sample. As explained above, beam scanningincreases eye safety, mitigates explosion risk, and averages themeasurement over possible sample inhomogeneities. The CCD frame time ofthe Raman spectrometer is chosen to be an integer multiple of thescanning period (also sometimes referred to as a “base period”), so thateach frame sees the same scanning pattern and experiences the samecorresponding signal variations. If this was not the case, each framemay see different signals, because (1) the optical system throughputvaries for different positions of the scanning mirror and (2) thedifferent spots illuminated on the sample may have differentreflectivities and/or chemical compositions, leading to differentspectrum intensities and shapes. Synchronizing the frame timing with thebeam scanning period makes it possible to suppress ambient light orsample fluorescence from the frames acquired with different Raman lasersas well as from potential background frames.

The ambient light sources may be modulated. For example, the intensityof ambient artificial light may vary at an integer multiple of the localutility frequency (e.g., 50 Hz or 60 Hz). Typically, a 100 Hz or 120 Hzmodulation of the ambient light is observed for indoor measurements. Toensure that the spectrometer measures a constant or nearly constantlevel of ambient light, one can set the frame period (i.e., the timebetween the start of two consecutive frames) to be an integer multipleof ambient light modulation period (also sometimes referred to as a“base period”). In some cases, this base period is based on a flickerperiod of an ambient light source (e.g., a light source powered by anelectric utility) that contributes to or is responsible for thebackground/ambient light. The base period can be an integer multiple ofthe flicker period, or vice versa. The flicker period of a known ambientlight source can be determined based on a frequency analysis of theambient light intensity, and can be (in some cases) about 8.33 ms toabout 10 ms, including all values and sub ranges in between.

In some cases, the flicker period can be automatically determined asfollows. The Raman system (i.e., any suitable Raman system as describedherein) can include a GPS receiver (e.g., see I/O 1434 in FIG. 14) thatcan receive GPS information and determine its own location as asurrogate indication of the location of the sample. Once the sample'sGPS location is known (e.g., the country of location), it can becompared against a table listing the electric current frequency employedby utilities in that location. For example, if the GPS locationindicates the location of the sample as the United States, the flickerperiod can be estimated based on the knowledge that the electricitystandard in the United states is 120 V, 60 Hz AC. Similarly, if the GPSlocation indicates the location of the sample as the United States, theflicker period can be estimated based on the knowledge that theelectricity standard in the United states is 220 V, 50 Hz AC. In othercases, user input can specify the geo-location of the sample (e.g., suchas via a user interface of the Raman system), and

Following these considerations, an example embodiment of a timingscheme/method is illustrated in FIG. 17, which shows the acquisitioncycle for differential Raman spectroscopy with interleaves measurementsduring frame periods selected based on beam scanning for eyesafety/explosion mitigation and ambient light modulation, and specifiesthat:

-   -   A frame period (also sometimes referred to as a “measurement        period”) is defined by a CCD integration time of 130 ms followed        by a readout time of 20 ms (the read-out time includes other        time buffers and delays for system operation). The frame period        (Laser 1 frame/Laser 2 frame/Background frame in FIG. 17) is        thus 130 ms+20 ms=150 ms.    -   A laser scanning system operates at 20 Hz along a slow axis and        200 Hz along a fast axis to illuminates the sample in a raster        scan pattern. The scan period is 1/(20 Hz)=50 ms. The frame        period (Overall exposure/acquisition period in FIG. 17) is thus        equal to 3 full scan periods.    -   The ambient light (Ambient Light Modulation in FIG. 17) is        assumed here to be modulated at twice the utility frequency        (either 50 Hz or 60 Hz based on the local standards). The light        modulation period is thus 1/(2×50 Hz)=10 ms or 1/(2×60 Hz)=8.33        ms. The frame period is thus equal to either 15 or 18 full light        modulation periods.    -   A measurement includes one or more of the following        sequence: (1) Laser 1 is turned on and illuminates the        sample; (2) the corresponding Raman spectrum is measured by the        CCD, integrating for 130 ms between t=0 and t=130 ms; (3) the        CCD is read out, laser 1 is turned off, and laser 2 is turned        on; (4) a second Raman spectrum is measured by the CCD        integrating for 130 ms, between t=150 ms and t=280 ms; (5) the        CCD is read out and laser 2 is turned off; (6) a third spectrum        (Background frame in FIG. 17) is measured by the CCD integrating        for 130 ms, between t=300 ms and t=430 ms; and (7) the CCD is        read out.

Such measurements result in three Raman spectra (i.e., a first Ramansignal/spectra, a second Raman signal/spectra, and a background/ambientlight signal/spectra) that can be used to extract the Raman spectrum ofthe sample (also sometimes referred to as a post-processed signature)and analyze its composition. The timing scheme described here is oneexample of how to synchronize the different modulations (ambient lightmodulation, laser beam scanning, spectrum acquisition frame). Othertiming schemes are also possible. As illustrated in FIG. 17, themeasurement of each Raman signal can be done during an integrationperiod (CCD integration signal in FIG. 17) within each frame. Saidanother way, the first Raman signal/spectra can be measured during afirst integration period, the second Raman signal/spectra can bemeasured during a second integration period, and the background/ambientlight signal/spectra can be measured during a third integration period,each of which can be equal to each other.

In choosing the same integration time for the frame with laser 1 turnedon and the frame with laser 2 turned on, it is assumed that the laserpower on target is similar for laser 1 and laser 2. This may not alwaysbe the case and can be accounted for by scaling the respective spectrato equalize the strength of the Raman and fluorescent signature or byvarying the integration times accordingly. In general, this results inthe presence of an ambient light signature of the subtraction of the twoRaman spectra. This signature may be unmixed using the information fromthe third frame (acquired with no laser on, measuring the ambient lightas reflected by the sample and collected by the system).

Polarized Difference Raman Spectroscopy

If the Raman pump laser is polarized, and a polarization analyzer isused on the collected Raman signal, different Raman spectra may berecorded for the different polarization states of the scattered Ramanphotons. In particular, the relative heights of the different Ramanpeaks may be different. Polarized Raman spectroscopy can provideinformation on molecular or crystalline orientation.

Polarization Raman information can be collected together with differenceRaman information. The Raman laser(s) output can be polarized prior toimpinging on the sample, and either using free-space coupling betweenthe laser and the telescope, or by using polarization-maintaining fiber,a polarized Raman pump beam can be sent to the sample (not shown). Apolarizer may also be used to polarize the unpolarized output of astandard multi-mode fiber, although this results in a loss of power. Onthe collection side, a polarizer, such as a tourmaline crystal, that canbe rotated between two orthogonal orientations can be used to analyzethe collected light. Polarization Raman information can be collected atboth wavelengths used in difference Raman spectroscopy, and thedifference would contain such information. Data analysis may includecomputing the difference between the polarized difference Raman spectraobtain at two different orientations of the analyzer.

FIG. 34 illustrates an example of how polarized difference Ramaninformation can be obtained by acquiring successive Difference Ramanspectra with two different orientations of a polarizer using anysuitable Raman system described herein. Raman spectra of a target sample3402 can be acquired using any suitable system as described herein, andincluding at least a Raman laser 3404 and a spectrometer 3406. Here, ina first setting 3400 a of the Raman system, a parallel polarizer 3408 ais employed in front of the spectrometer 3406, so that the spectrometerreceives a parallel polarized Raman signal. The collection protocol 3410a for this signal can be similar to that generally described for FIG.17, and include a) Raman spectrum acquisition at a first pumpwavelength; b) Raman spectrum acquisition at a second pump wavelength;and c) optionally, Raman spectrum acquisition with the laser(s) turnedoff. The result is a parallel-polarized Raman spectra of the sample3402.

Then, in a second setting 3400 b of the Raman system, a cross polarizer3408 b is employed in front of the spectrometer 3406, so that thespectrometer receives a cross polarized Raman signal. The collectionprotocol 3410 b for this signal can also be similar to that generallydescribed for FIG. 17, and include a) Raman spectrum acquisition at afirst pump wavelength; b) Raman spectrum acquisition at a second pumpwavelength; and c) optionally, Raman spectrum acquisition with thelaser(s) turned off. The result is a cross-polarized Raman spectra ofthe sample 3402. The difference between the parallel-polarized Ramanspectra and the cross-polarized Raman spectra can be computed at 3412 asthe polarized difference Raman spectra.

Camera and/or Eye-Piece

The system may include a camera or an eyepiece that images the samplewith enough magnification to distinguish features associated with thesample texture or granularity. This allows the user to locatepoints-of-interest, such as isolated particles to be analyzed. Thecamera may be used to add a functionality like that of a digitalmicroscope to the Raman system.

In one embodiment, the Raman system has a fixed focal distance (e.g.,5-15 cm) and is positioned vertically, pointing down, with a horizontalfocal plane. The user may position a sample, for example, using athree-axis stage, to bring an area of interest, for example, a particleor a fiber, at the Raman laser focus. The visual feedback provided bythe instrument camera or eye-piece may be used to assist the user inthis task. Once the sample is positioned properly, a measurement can beinitiated, and the instrument can analyze the resulting spectralinformation to determine the composition of the analyte (e.g., aparticle or fiber on a substrate). In another configuration, the Ramaninstrument is mounted on a vertical translation stage and the sample ismoved using a two-dimensional translational stage in the horizontalplane. Overall, this provides three degrees of freedom for positioningthe sample at the focus of the Raman laser.

In contrast to existing Raman microscopes, the instrument described hererequires no ambient light shield, yet still offers increased eye safety,even in the absence of an enclosure around the entire instrument oraround the sample compartment. This could allow for an open design thatmakes it easier to manipulate the sample. Since the increased eye safetyuses laser beam scanning over an area about 1 mm×1 mm, the spatialresolution may be limited. It may sometimes be desirable to interrogatea smaller object (e.g., a particle with <100 μm diameter). In such acase, one can turn off the laser beam scanning. An interlock can thenprevent the lasers from turning on until a sample compartment orinstrument enclosure is closed. This interlock prevents the instrumentfrom emitting an accessible beam whose effective irradiance is higherthan that of a scanned beam. The instrument may thus maintain the lasersafety classification obtained with the laser beam scanning on. Forexample, a class 3R Raman microscope may be designed in such manner.

A difference Raman system, paired with the microscopy capability, allowsanalysis of a sample with strong fluorescence and samples deposited onfluorescent substrates.

The standoff capability of the instrument described here, when pairedwith the imaging capability, results in a Raman microscope with a longworking distance, which may be advantageous when analyzing a bulkysample.

Coarse Imaging

Forming small images of a target analyte can increase sensitivity andspecificity for the analysis of solid mixtures and provide anapproximate quantification from the statistical analysis of the particledistributions.

Consider a solid mixture with a dominant compound containing a smallquantity of a minority compound. This may be a pharmaceutical pill witha small quantity of active compound embedded in a matrix of an inactiveexcipient. In the limit of uniform mixing of very small crystals, allsub-volumes of the sample have the same composition. However, if thegrain sizes of the different compounds have a certain minimum size, andif small volumes whose dimensions do not greatly exceed the dimensionsof the grain size in the mixture are probed, different volumes may havedifferent concentrations of the minority compound. Thus, it may beadvantageous to probe several distinct volumes to increase the odds offinding a volume with a larger concentration of the minority compound.This is one rationale for scanning a sample to increase the limit ofdetection of a minority compound.

Coarse imaging may also be useful to assess the uniformity of the mixingof different compounds in a powder mixture or pill, e.g., by measuringthe relative concentrations of the different compounds in severaldistinct volumes. A spatial map can be created, revealing the spatialdistribution of the different compounds and potential clusteringeffects.

Image Stabilization

The operator's hand motion can result in a drift of the instrument aimpoint during a measurement. If the sample is large, such as a bulkamount of powder, a useful measurement may be acquired even with pooraiming stability. However, if the sample is small (e.g., a grain orparticle), poor or varying aim may result in a very inconsistentmeasurement, with varying mixing ratios between sample and substratesover the duration of the measurement. Proper analysis or chemicalidentification may then take longer.

A camera can be used to mitigate errors caused by drift in theinstrument's aim point due to the operator's hand motion. Morespecifically, the camera can lock the aim point based on visual cues.Examples of visual cues are sample texture, topographical features,particles, and defects. The camera image may provide the input for animage stabilization system that relies on a beam steering mechanism tocompensate drift of the aim point: (1) the camera acquires a firstimage, (2) the camera acquires a second image, (3) the onboardelectronics analyze the two images are analyzed to approximate theangular deviation that could correct for a possible shift between thetwo images, and (4) the onboard electronics update the position of alaser beam scanning mirror to deflect the instrument aim by an amountopposite to the angular deviation observed. This compensates for theangular deviation. The process is then repeated as desired. This controlloop uses two consecutive images as input and outputs a drive signal toa laser beam steering system. This implies that the optical path for theimaging system passes through the beam steering optics.

Alternatively, the input to the control loop may be the position of alaser spot within an image, e.g., with respect to some feature in theimage (e.g., a particle of a given color or shape). If the distancebetween the sample and the instrument is known, the distance between twopoints on an image can be converted to an angular deviation as seen fromthe instrument output. That angular deviation can be used to produce thecontrol signal to the beam steering optics, allowing compensation foraim fluctuations. This method does not require the optical path for theimaging system to pass through the beam steering optics.

The system may allow the user to indicate a feature of interest ordesired measurement spot, for example, by pressing a touchscreendisplaying an image of the area surrounding a sample, on which thesystem can lock. For example, the system may automatically steer thetelescope optics to focus the Raman probe to that spot. The systemmaintains that aim, correcting automatically for the hand movements ofthe operator using information from the camera.

Focus Adjustment/Autofocus

The system may include a degree of freedom in the optical sub-system toadjust the distance at which the Raman lasers are focused. In otherwords, the standoff distance may be adjustable, either manually or bythe operation of a motorized actuator. Examples of standoff distancetuning ranges may be from 30 cm to 4 meters, from 10 cm to 120 cm, from2 cm to 25 cm.

Since the Raman laser beam sent to the sample and the Raman scatteringcoming from the sample follow the same path (i.e., the Raman scatteringtraces the laser beam's path backward through the telescope), if thesystem focuses the Raman laser beam on the sample, then the collectionof the Raman scattering is also typically optimized.

FIGS. 18A and 18B illustrate the use of visible laser beams 1810 to markthe Raman pump beam's focus 1870 in a Raman spectroscopy system with avariable focus telescope/movable lens 1820 and a dichroic beam combiner1830 that couples the visible laser beams and the Raman pump through thesame optics. Crossing laser pointers may be used to indicate where thefocal plane of the instrument is located: two visible laser beams 1810,collimated or with a low divergence, having a small diameter (forexample between 0.5 mm and 5 mm) can be made to intersect at a point1870 that is both on the optical axis of the instrument and in the focalplane of the instrument. Ignoring dispersion in the optical system, thatpoint 1870 is thus where the Raman laser is focused. One of the twobeams may be colinear with the instrument optical axis, so that italways indicates where the instrument is aiming, even if the sample isnot in the focal plane.

The two beams may be differentiated with different colors. The two beamsmay illuminate the sample continuously or they may be pulsed. Suchblinking laser pointers may be easier for the human eye to distinguishin bright illumination conditions. The brightness of the laser pointersmay be tuned based on the ambient light brightness, e.g., based on usercomfort. A sample fluorescence, pumped by the visible laser beam(s), maybe detectable by the instrument. It may thus be desirable to turn on thevisible pointer lasers selectively in-between CCD integration periods,for example during the read-out time between two consecutive frames.

As the instrument focal length or working distance may be adjustable, itis desirable that the directions of the two visible beams 1810 areadjusted accordingly, so that the two beams always intersect where theRaman laser is focused. We describe here one engineering solution tothis issue: two visible laser beams, collimated or with a lowdivergence, having a small diameter (e.g., 1 mm diameter) are generatedinside the instrument. Both beams are overlapped with the Raman laserbeam using a dichroic beam splitter/mirror 1830 that is mostlytransmissive for the Raman laser wavelength and mostly reflective forthe wavelength of the visible laser beams. The visible beams are alignedso that they propagate parallel to the Raman laser beams at a point inthe optical system where the Raman beam is collimated. The dichroic beamsplitter is positioned within the optical system such that the visiblebeams pass through the moveable optical components (here, the movablelens 1820) of the optical system before exiting the instrument.

A first visible beam propagates along the optical axis at all pointsalong its path. As a result, this beam indicates where the instrument isaiming. The second visible beam propagates parallel to the optical axis,but is decentered with respect to the optical axis, such that it comesout of the instrument at the edge of the optical aperture. Since thisbeam follows a path like the marginal rays in the optical instrument, itintersects the optical axis at the point where the instrument isfocusing the Raman laser beams. We assume here minimal dispersion suchthat the focal point of the instrument is mostly similar at thewavelength of the visible beams and at the wavelengths of the Ramanlasers. Since this second visible beam follows the trajectory of amarginal ray for the Raman lasers, and this remains true regardless ofthe position of the moveable components within the optical system, thesecond visible beam intersects the optical axis at the point where theinstrument is focusing the Raman lasers. Consequently, the intersectionof the two visible beams remains coincident with the focus of the Ramanlasers, even as the instrument focal length is varied.

The dichroic beam splitter may also be inserted at a point where theRaman beam is not collimated (e.g., after the beams 1810 pass throughthe lens 1820). In this case, the on-axis pointer laser should still beparallel and coincident with the optical axis. And the second laser beamis inserted such that its direction after reflection from the dichroicbeam splitter is parallel to the direction of the Raman ray crossing theoff-axis position where the second laser pointer is inserted.

The system may include an autofocus mechanism to automatically adjustthe distance between the instrument and the location of the Raman focus.This makes it easier to place the Raman focus is located on or in thesample. The autofocus may operate during the measurement to compensatefor possible hand movements during the measurement. The autofocus systemmay allow for the definition of an offset such that the system isfocusing some distance away from the first scattering surface, or somedistance away from the surface that scatters light most. For example,the sample may be a liquid in a semi-transparent container: theautofocus feedback signal may indicate the distance from the instrumentto the first scattering surface, in this case the interface between airand the semi-transparent container. But the system should be focusedinside the liquid sample for adequate measurement of the analyte. Anoffset setting may thus be used so that the system focuses a shortdistance behind the first scattering surface, for example, 1 cm behindthe first scattering surface. That offset may be set by the user orselected automatically. To implement this offset, the autofocus feedbacksignal uses a local ranging capability, i.e., the capability to measurethe position of the system focal point with respect to the mainscattering surface.

The autofocus mechanism may use a camera, such as the camera 1840, thatlooks out at the scene facing the instrument. The camera is displacedlaterally a certain distance from the telescope optical axis. Theon-axis laser pointer illuminates one point 1850 on the scene, and theposition of that point with respect to the camera optical axis 1860gives a rough estimate of the distance between the object illuminated bythe on-axis laser and the instrument. This method is generallyclassified as a parallax method of estimating the rage of an object.This calculation exploits the known, constant distance and orientationof the Raman telescope optical axis with respect to the camera opticalaxis. The relationship between the position of the image the on-axislaser pointer spot, as seen by the camera 1840, and the distance fromthe instrument to that spot may be calibrated beforehand (e.g., in afactory) and stored as a lookup table.

A finer autofocus mechanism may be used after adjusting the position ofthe different moving elements of the Raman telescope according to thatrange information. This may rely on imaging the two spots from the twolaser pointers and ensuring that they are overlapped. The relativeposition of the spots formed by the two laser pointers intersecting withthe main scattering surface is a directional measure of the distancebetween the main scattering surface and the position of the Raman focus.It can thus be used as an input to a closed-loop control of the focusposition.

FIG. 19 illustrates such use of a camera system for imaging and trackinga standoff Raman system's aim point. In FIG. 19, two cameras (camera 1,camera 2), with camera 1 having the same axis as the optical setup andcamera 2 being offset from the optical setup, are employed. For camera1, the image of laser 2 is always formed on its optical axis and in thecenter of its corresponding image, while for camera 2, the image oflaser 2 is always formed off its optical axis. When laser 1, laser 2(sometimes collectively referred to as a “ranging laser”) intersect atthe position of Raman focus (Position B), the images of these lasers arecoincident with the optical axis of camera 1, and are equally spaced(but coincident with each other) from the optical axis of camera 2. ThePosition A, Position B images of camera 1 and camera 2 both indicates anoffset between the images of laser 1, laser 2, indicating that thesepositions are not at the Raman focus. A user can then manipulate amovable element (such as the movable lens of FIGS. 18A, 18B) to obtainthe desired Position B images for laser 1, laser 2.

If an offset is desired between the main scattering surface and themeasurement spot (where the Raman laser is focused), the close-loopcontrol can be set to target a specific non-zero distance (denoted as Δxin FIG. 19) between the spots formed by the two laser pointersintersecting with the main scattering surface. The Raman laser/pump beamthen gets focused to a spot on or just below the sample surface. Thedistance between the focus of the Raman laser/pump beam and the Ramansystem itself can be about 2 cm, about 6 cm, about 10 cm, about 30 cm,about 90 cm, about 400 cm, including all values and sub ranges inbetween.

Accordingly, in some embodiments, a method of Raman spectroscopy, as canbe carried out with any Raman system described herein, can include thesystem measuring the space between the Raman system and the sample, suchas the sample of FIG. 19. In some cases, the spacing can be about 2 cm,about 10 cm, about 100 cm, about 400 cm, including all values and subranges in between. This measurement of the range of possible distancesbetween the sample and the Raman system can be done, in some cases, bydetecting the spot generated by the laser 2 (along the optical axis)using camera 2 (off the optical axis), and estimating the range based onthe image of that spot on camera 2. In some cases, the distance betweenthe spots formed by laser 1 and laser 2 on camera 1 and/or camera 2,detected by acquiring a sample image, can be used to make themeasurement as explained herein. The autofocus mechanism can thenoperate to reduce the spacing/distance between the spots to make themoverlay each other. Once done, the sample can be illuminated with theRaman pump beam, and the Raman signal/spectra measured.

Depth Profiling

The system may be capable of locating the position of the differentinterfaces along the optical axis. For example, if the instrument istargeting an analyte such that several transparent or semi-transparentobjects (e.g., glass or plastic walls, windows, enclosures, containers,etc.) are positioned between the instrument and the analyte, theinstrument may be capable of measuring the distance between theinstrument and the successive interfaces. This can be achieved, forexample, using a time-of-flight ranging system. The system may thenprompt the user where the instrument should be focused with respect tothese interfaces. For example, if an analyte is contained in a glasscontainer located behind the closed window of a fume hood, theinstrument should be focused a short distance (e.g., 5-10 mm) behind theinterface formed by the air and the glass container. The instrument maybe capable of autonomously and sequentially probing several points at ornear the interfaces observed.

Uses of a Handheld Difference Raman Spectroscopy System

A handheld standoff (or proximal) difference Raman instrument canidentify an unknown chemical by acquiring its Raman spectrum andcomparing the Raman spectrum to a library of known Raman spectra. It canbe used by pointing the instrument toward a sample located a shortdistance away from the instrument and pressing a trigger or other switchto initiate a measurement. The system then illuminates the sample with aRaman laser, collects the Raman scattering, obtains a spectrum of theRaman scattering using a spectrometer, digitizes the resulting spectrum,and uses various processing analogous to linear regression processing tocompare the measured spectrum to a library of known spectra. For theinstrument described here, several spectra can be used as input to theprocessing, including Raman spectra acquired at different Ramanwavelengths and background spectra acquired with no Raman laser turnedon.

The system may have a standoff distance that allows measurement throughdifferent interfaces (e.g., windows, enclosures, container walls). Thestandoff distance also allows measurement in cases where the instrumentcannot be placed in proximity to a sample, either because a physicalbarrier exists, or because there is a risk of contamination to theinstrument and/or user, or because any other ambient condition mayprevent the instrument from being safely or practically placed near thesample.

The difference Raman system described can be used to analyze:

-   -   Fluorescent samples, samples placed on fluorescent substrates,        and samples in containers with fluorescent walls;    -   Absorbing, dark, or colored materials, that may present an        ignition risk;    -   Hot materials that may emit a strong blackbody radiation;

In addition, materials that can be readily measured by most existingRaman instruments can be measured by the instrument described here.

The instrument may be separated into two parts: (1) a telescope probecontaining the optical system that focuses the Raman light onto thesample, collects the Raman scattering, and performs desired auxiliaryfunctions (e.g., motor and associated controller to adjust the telescopefocal distance, camera to visualize the sample or the scene around thesample, camera used for autofocus or image stabilization, laser pointersto indicate aim and focus position, laser beam scanning system for eyesafety and explosion risk mitigation); and (2) an instrument core,containing the lasers, the spectrometer, and most of the electronics,interfaces, antennas and power supply. The two parts may be linked by anumbilical cable containing both electrical conductors for power andcontrol signals as well as optical fibers to send the Raman laser lightto the telescope and the Raman scattering back from the telescope to thespectrometer.

FIG. 16 illustrates such an example Raman system 1600 with the probeseparated from the instrument. It is understood that, unless explicitlynoted otherwise, components of FIG. 15 may be functionally and/orstructurally similar to similarly named components of FIGS. 14, 15. Forexample, the telescope 1608 may be similar to the telescope 1508, and soon. FIG. 16 illustrates how the probe 1604, telescope 1608, camera 1610,motor 1630, and the motor controller 1628 can be disposed in acompartment/casing 1601 b of the system 1600, while all other componentscollectively reside in a separate compartment 1601 a.

Explained with reference to FIGS. 14-16, but applicable to any Ramansystem described herein, in some cases, a dispersive element may bedisposed in the optical path prior to the spectrometer (e.g., betweenthe probe 1404 and the spectrometer 1412) that receives the Raman signaland/or the background ambient signal. In this manner, the Ramansignal(s) (e.g., the first Raman signal from laser 1 and the secondRaman signal from laser 2 in FIG. 16) and/or the background/ambientlight signal can be dispersed into their respective spectral components.Then, a detector array, such as the CCD 1414 in some cases, may beuseful for detecting the spectral components to generate thecorresponding spectra.

A display may be integrated with the instrument, with switches andbuttons allowing the user to control the instrument. Alternatively, aremote controller, potentially with a screen, can be used as a userinterface. Communication between the instrument and the remotecontroller may be via wired or wireless interfaces.

The standoff difference Raman spectrometer may be integrated onto arobotic platform (e.g., an unmanned ground or aerial vehicle).

A complete standoff difference Raman spectrometer or the telescope partof a two-part system may be mounted on a gimbal system. This gimbalsystem can be used to adjust the laser beam's orientation and to aim thelaser beam at specific objects or analytes.

Reaction Monitoring

The system may be used to monitor a chemical reaction. Different probescan be used for such an application. A Raman probe may be directlycoupled to a flow cell, or via one or more obstructions. FIG. 20illustrates an example handheld standoff difference Raman spectrometerthat focuses a Raman pump beam onto an analyte through obstructions,including a window and a wall of a container, for monitoring a chemicalreaction in a beaker. (The spectrometer of FIG. 20 can be used for othermeasurements as well.) As illustrated in FIG. 20, in such setups, thewindow and/or the walls of the container can generate Raman scatteringsignals, so a user can choose a focus position for the Raman beam basedon these scattering signals. A real-world example of such a setting isshown in FIG. 37, where a handheld standoff Raman system 3700 is usedfor identifying a chemical compound in a container 3710 through theglass window 3720 of a fume hood 3730, and from a distance of more thanone foot.

A telescope probe, as described in the two-part system above, may beused to focus the Raman laser into a reaction vessel. The measurementcan be realized either through the walls of the vessel, if the vessel ismade of glass or plastic, or through a transparent observation port oran opening of the vessel.

FIG. 21 illustrates an example two-part standoff differential Ramanspectroscopy system, controlled with a wireless controller 2110, with aprobe module 2120 mounted on a robot arm 2130 and a core module 2140(e.g., which can contain one or more other components as described forFIGS. 14-16) mounted on the robot chassis 2150 for testing a sample2160.

FIG. 22 illustrates a two-part standoff differential Raman spectroscopysystem useful in a laboratory setting for testing samples on microscopeslides, for example. FIG. 22 illustrates the system, controlled with awireless controller 2210, with a core module 2220 and a probe module2230 mounted on a vertical stage 2240 above a sample stage 2250.

FIG. 23 illustrates a two-part standoff differential Raman spectroscopysystem useful for testing samples through an opening in a vesselcontaining the sample, for example. FIG. 23 illustrates the system,controlled with a wireless controller (not shown), with a core module2310 and a probe module 2320 mounted on a fixed ring holder 2330 above asample 2340.

FIG. 24 illustrates a two-part standoff differential Raman spectroscopysystem useful for testing samples through a side wall in a vesselcontaining the sample, for example. FIG. 24 illustrates the system,controlled with a wireless controller 2410, with a core module 2420 anda probe module 2430 mounted on a fixed ring holder 2440 to the side of asample in a beaker 2450. FIG. 36 is a photograph of a one-part variationof the two-part system of FIG. 24, and shows a one part Raman systembeing similarly used to monitor a reaction inside a vessel. Any Ramansystem described herein, when employed in such a setting, can analyzethe solution and determine in real-time its chemical composition,including reactants, products and intermediates.

FIGS. 25A-25B illustrates a two-part standoff differential Ramanspectroscopy system with adjustable working distance, which can besimilar to the system of FIG. 23 and be useful for testing differentmaterials/phases in a sample through an opening in a vessel containingthe sample. FIG. 25A shows the system focusing on a first liquid phase(Material 1) of the sample in a beaker, while FIG. 25B shows the systemfocusing deeper into the sample and onto the second liquid phase(Material 2), in order to measure the Raman spectrum for one or theother phase. The vertical position of the instrument could be adjustedusing an external mechanical device (not shown) instead of adjusting theworking distance of the system with the internal optics of the system.

FIGS. 26A-26B illustrates a two-part standoff differential Ramanspectroscopy system useful for testing different materials/phases in asample through a side wall in a vessel containing the sample, such as ina chemistry glassware. FIG. 26A shows the system focusing on a firstliquid phase (Material 1) of the sample in a beaker, while FIG. 25Bshows the system focusing at a different height into the sample, andonto the second liquid phase (Material 2), in order to measure the Ramanspectrum for one or the other phase. The position and aim of theinstrument can be adjusted using an external mechanical device (notshown) in order to measure the Raman spectrum for one or the otherphase. Such a mechanical device may be manually actuated or motorized.

Some advantages of the system described here for such application are:mitigation of fluorescence (using difference Raman techniques),mitigation of ignition or explosion risk (via laser beam scanning),ability to operate with no ambient light shield (enabled by theacquisition of rapidly interleaved background frames and proper timingof the different processes), ability to measure at a distance, throughwindows, container walls or other obstacles (enabled by the standoffmeasurement modality).

The system may be used to monitor the relative concentrations ofdifferent species in a mixture, for example, reaction reagents,intermediate products, and final products. The system may output a graphof the relative concentration of the different compounds as a functionof time.

Even if unknown compounds are present, the system's electronics may beable to unmix the measured total spectrum to obtain the spectra of thedifferent compounds contributing to the total spectrum. Such unmixingbecomes more reliable as system measures different concentration ratiosfor the different compounds over time.

The instrument's onboard electronics may also be able to track specificpeaks in the Raman spectrum, potentially associating these peaks withspecific chemical groups, and inferring possible chemical structures forthe unknown compounds being measured.

When a measurement is realized through the walls of a reaction vessel,possible condensation on the walls of the reaction vessel may be aconcern. Condensation may form if the vessel and/or its content iscooled to a temperature below the dew point of the ambient air.Condensation results in increased scattering of the Raman pump beam andof the Raman signal, decreasing the signal throughput and thesignal-to-noise ratio of the measurement. To mitigate this issue, twomain strategies may be used:

-   -   (1) the air immediately surrounding the external wall of the        vessel may be maintained dry enough that the dew point is kept        at a lower temperature than the temperature of the external        wall. This may be achieved by enclosing the vessel in an        enclosure filled with a dry gas, in which case the Raman system        may be in or out of the enclosure. Alternatively, a flow of dry        gas may be guided along the external walls of the reaction        vessel.    -   (2) The temperature of the external wall of the reaction vessel        may be maintained high enough that it is above the dew point of        the ambient air. This may be achieved by thermally isolating the        external wall from the solution contained in the reaction        vessel, for example using a cavity or jacket filled with a low        heat conduction material or vacuum. This thermal isolation        solution should be transparent at the wavelength of the Raman        pump laser and over the spectral range of the desired Raman        signal. For example, a closed glass jacket filled with vacuum        would provide such solution.

FIGS. 38A-38B illustrates an example approach to prevent or reduceformation of condensation, and illustrates an example reaction vessel3803 being probed by a Raman system 3801. A standoff Raman analyzer 3801analyzes the composition of a solution 3802 inside the reaction vessel3803, and the condensation could otherwise lower the signal obtainedwhen the Raman system 3801 analyzes the composition of the solution3802. The vessel 3803 has a wall with cavities 3804 and 3805. Cavity3805 allows for circulation of a temperature regulation material 3806(such as any suitable liquid, or gas) to control the temperature of thesolution 3802. The temperature regulation material exits at 3807. Cavity3804 is either closed (as illustrated in FIG. 38B) or allows for thecirculation of a liquid or gas (as shown in FIG. 38A). The materialcontained in or circulated through 3804 can either be a dry gas, vacuum,or other dry, low heat conduction material that allows for transmissionof light in the visible and near-infrared spectral range. The reactionvessel is optionally closed with a lid 3810 or other interface. In theabsence of cavity 3804, the external wall of the reaction vessel 3803may be cold and condensation may form, scattering the Raman pump beamand the Raman signal and decreasing signal throughput. The vessel 3803shown here may maintain the temperature of its external wall above thedew point, avoiding the formation of condensation.

FIGS. 39A-39B illustrate another example approach to prevent or reduceformation of condensation and illustrates a dry enclosure 3908 used toprevent formation of condensation on the walls of a reaction vessel 3903disposed therein. The standoff Raman system may be located inside oroutside the dry enclosure. A standoff Raman analyzer 3901 analyzes thecomposition of a solution 3902 inside a reaction vessel 3903, and anycondensation could lower the signal obtained by the Raman system. Thevessel 3903 may have a wall with cavities 3904 allowing for circulationof a temperature regulation material 3905 to control the temperature ofthe solution 3902. The temperature regulation material exits at 3906.The reaction vessel in contained in an enclosure 3908 filled a dry gasor other dry, low heat conduction material that allows for transmissionof light in the visible and near-infrared spectral range. The reactionvessel is optionally closed with a lid 3907 or other interface. TheRaman analyzer 3901 may be outside the dry enclosure (as illustrated inFIG. 39A) or inside the dry enclosure (as illustrated in FIG. 39B). Inthe absence of the dry enclosure 3908, condensation may form on theexternal wall of the reaction vessel 3903, scattering the Raman pumpbeam and the Raman signal and decreasing signal throughput. The dryenclosure 3908 lowers the temperature of the dew point, avoiding theformation of condensation on the walls of the reaction vessel 3903.

FIG. 40 illustrates another example approach to prevent or reduceformation of condensation and illustrates a dry gas flow used to preventformation of condensation on the walls of a reaction vessel that theflow interacts with. A standoff Raman analyzer 4001 analyzes thecomposition of a solution 4002 inside a reaction vessel 4003, and anycondensation could lower the Raman signal thereby obtained. The vesselmay have a wall with cavities 4004 allowing for circulation of atemperature regulation material 4005 to control the temperature of thesolution 4002. The temperature regulation material exits at 4006. A drygas 4008 or other dry, low heat conduction fluid that allows fortransmission of light in the visible and near-infrared spectral rangemay flow along the walls of the reaction vessel 4003 to avoidcondensation on the walls. The reaction vessel is optionally closed witha lid 4007 or other interface. In the absence of dry gas 4008,condensation may form on the external wall of the reaction vessel 4003,scattering the Raman pump beam and the Raman signal and decreasingsignal throughput. The dry gas 4008 lowers the temperature of the dewpoint, avoiding the formation of condensation on the walls of thereaction vessel 4003.

Robot-Mounted System

A standoff differential Raman spectroscopy system may be mounted on arobotic platform (air, ground or water-based), either remotelycontrolled or autonomous, as generally described for FIGS. 21, 27.Several features of the standoff difference Raman instrument make itwell suited for such robot-mounted applications:

-   -   The standoff distance allows to maintain a safe distance between        the robot and the analyte, reducing the risks of collisions        between the robot and objects surrounding the analyte        (containers, furniture, other analytes, etc.);    -   The standoff distance also allows to measure through obstacles        such as clear container walls or windows;    -   The adjustable working distance makes it convenient to focus the        system onto the analyte without requiring fine position control        of the robot;    -   The visible laser pointers give feedback on the system aim and        focus;    -   The range information (parallax) from the laser pointers give        situation awareness to the robot controller and can prompt him        to adjust the robot position for the measurement;    -   The camera image allows the user to see where the system is        aimed and focused, and it allows to gather more information on        the analyte (shape, color, etc.).

The standoff differential Raman spectroscopy system can be controlledremotely, either through a dedicated wired or wireless communicationlink or by interfacing with the robot communication system.

A modular standoff differential Raman spectroscopy system may beconverted by a user from a handheld system to a robot-mounted system.FIG. 27 illustrates a modular standoff differential Raman spectroscopysystem that can be converted by a user from a handheld system 2710 to arobot-mounted system 2750. Such system features a core component 2720common to both modalities, and a set of adaptor plates (2730, 2740)containing the features particular to one modality or the other. Forexample, a handle 2730 may be desired for a handheld system but it isnot required for a robot-mounted system. The handle 2730 can bedetachable. Alternatively, a robot-mounted system may have requirementsin term of mechanical, electrical, and communication interface with therobot that can be captured by a specific adaptor plate, such as theplate 2740.

Macroscope Embodiment

FIGS. 28A-28C illustrate Raman macroscope embodiments, offering both avisible image of the sample and enabling to run a Raman analysis ofspecific points within the field of view. The working distance isbetween 1 cm and 20 cm. Different mounting brackets and/or other optionsare presented: a stand-alone system 2810 held in place usingindependently adjustable/retractable feet (FIG. 28A); a tripod mountedsystem 2820 using a standard ¼-20 threaded hole or other mechanicalinterface (FIG. 28B); macroscope or stereo-microscope mount withincluded height adjustment (FIG. 28C). Mechanical interface can use thestandard 3″ cylinder mount of most commercial macroscope mounts.

The system may include a housing (that may in turn include a mountingbracket/options as noted herein) and be assembled with an opticalfront-end/assembly that allows for shorter working distance, between 2cm and 20 cm for example. A short working distance while maintain alarge optical aperture (e.g. between 1″ and 2″) can allow to collect theRaman signal with a large numerical aperture, which results in largecollection efficiency. The optical front-end can also be used to imagethe sample at shorter wavelengths (e.g. in the visible spectral range)onto a camera (e.g. CCD or CMOS sensor).

Since it is in practice difficult to design an optical system with bothhigh object-side numerical aperture and large field of view, it may bedesirable to keep the field of view of the Raman optical subsystemsmall, and to allow for motion to bring the Raman beam over differentpoints to be analyzed. In some cases, the camera can image the sampleover a field of view greater than 5 mm with an object-space numericalaperture of less than 0.2, when the optical assembly has an object-spacenumerical aperture of at least 0.3 and a field of view of less than 2mm. The motion can be either: (1) motion of the optical head withrespect to a fixed instrument, with a fixed sample; (2) motion of thesample with respect to the fixed instrument and fixed optical head; (3)motion of the instrument (including optical head) with respect to afixed sample.

The motion can be manually driven or motorized, using a motioncontroller. This allows to bring the high numerical aperture Raman beamabove specific points to be interrogated, instead of relying on anoptical design with both a high numerical and a large field of view forthe Raman optical subsystem. The high numerical aperture (e.g. NA>0.2)of the Raman optical subsystem is desired to allow for larger collectionefficiency and consequently larger signal. By opposition, the visibleoptical subsystem can have lower numerical aperture (e.g. NA<0.2), sincethe camera sensor (CCD, CMOS) can be very sensitive and can produce goodquality image, which makes obtaining a large field of view (e.g. 1″×1″)for the visible optical subsystem more practical. Both the Raman opticalsub-system and the visible optical sub-system can have independentlyadjustable focus to allow their respective beams (i.e., the Raman beamand the visible beam) to come to focus at the sample surface or on aparticular point of interest, which can be automatically identified bythe processor/CPU of the system. By varying the focus of the visibleimage, a z-stack image can be acquired, with a 3D representation of thesample surface. This can be used to identify and sort the location ofthe points to be analyzed.

FIG. 29 illustrates a possible result screen (e.g., acquired by thesystem of FIG. 28B, and showing a fingerprint on a car door handle),showing a visible image 2910 of the area of interest, captured with acamera operating in the visible spectral range, and the results ofRaman-based chemical identification realized at a discrete set of pointsacross the area of interest. The locations of these points (indicated inFIG. 29 as the areas corresponding to “No match”, “RDX”, “Sugar”, etc.)can be decided automatically by an image analysis algorithm. Forexample, particles to be analyzed can be located on the image based onbrightness, shape (boundaries), polarization response (if polarizedlight is used for illumination and/or a polarization analyzer is used infront of the camera) or other methods. Random sampling or systematicanalysis of all points located on a predefined grid or set of points canalso be realized.

FIG. 30 illustrates several industrial design features of the macroscopesystem, including retractable or foldable feet 3010 that can possibly beindependently adjusted to vary the angle of regard of the system; athreaded hole 3020 as a mechanical interface, for example for tripodmounting; shock absorbers to provide ruggedness to the system andprevent damages in case of drop; and a macroscope mechanical adaptor,constituted for example by a cylinder with ˜3″ outer diameter to fitmost existing macroscope or stereoscope mounts.

Such macroscope would contain similar components as the ones containedin the handheld system described above, with the possible addition of amotion controller and mechanical stages allowing for the translation ofthe optical head with respect to the fixed instrument body. The opticalhead may also be modified to improve the image quality for the visibleoptical sub-system and the numerical aperture for the Raman opticalsub-system. Accordingly, FIG. 31 illustrates an example system diagramof such a macroscope 3100. Unless noted otherwise, components of themacroscope 3100 may be structurally and/or functionally similar tosimilarly named components in FIG. 14-16. The system 3100 includes amoving subassembly 3100 a (sometimes also referred to as the “opticalhead”) that includes the probe 3104, the camera 3110, and the requisitebeam shaping/guiding optics or optical assembly (MEMS mirror 3140, NIRfocusing optics 3142, visible light focusing optics 3144, a beamsplitter 3146, and visible-NIR focusing optics 3148 for optical couplingto the sample 3106. The Raman laser source 3102 and the spectrometer3112 are formed outside the moving subassembly 3100 a. An LEDilluminator 3160 illuminates the sample to allow the acquisition ofbright enough images with the visible imaging system.

The system 3100 also includes a motion controller 3150 and mechanicalstages (not shown) allowing for the translation of the optical head 3100a with respect to the (rest of the) fixed instrument body. A flexiblebellow 3170 allows the optical head to move with respect to the systemenclosure while allowing the system to remain sealed. The motion can bemanually driven or motorized, using the motion controller 3150. Thismakes it possible to bring the high numerical-aperture Raman beam abovespecific points to be interrogated, instead of relying on an opticaldesign with both a high numerical and a large field of view for theRaman optical subsystem. The high numerical aperture (e.g., NA>0.2) ofthe Raman optical subsystem is desired to allow for higher collectionefficiency and consequently larger signal.

In contrast, the visible optical subsystem can have lower numericalaperture (e.g., NA<0.2), since the camera sensor (CCD, CMOS) can be verysensitive and can produce good quality image, which makes obtaining alarge field of view (e.g., 1″×1″) for the visible optical subsystem morepractical. Both the Raman optical sub-system and the visible opticalsub-system (the optics 3142 and the optics 3144) can have independentlyadjustable focus to allow their respective beams to come to focus at thesample surface or on a particular point of interest. By varying thefocus of the visible image, a z-stack image can be acquired, with a 3Drepresentation of the sample surface. This can be used to identify andsort the location of the points to be analyzed.

FIG. 32 illustrates a system 3200 having a subassembly 3200 a that canbe similar to the system 3100, subassembly 3100 a respectively in FIG.31. In the system 3200, the motion controller 3250 controls movement ofa motorized sample stage 3250, instead of (and/or in addition to)movement of the subassembly 3200 a such as via, for example, control ofan actuator.

FIG. 33 illustrates a system 3300 having a subassembly 3300 a that canbe similar to the system 3100, subassembly 3100 a respectively in FIG.31. In the system 3230, the motion controller 3350 is disposed externalto a casing of the system 3300, and controls collective movement of thesystem 3300, including the subassembly 3300 a, with respect to astationary sample such as via, for example, control of an actuator.

It is understood that the various embodiments disclosed herein can becombined to obtain additive and/or alternative functionality. As anon-limiting example, the approaches to monitoring temperature riseand/or the use of fail-safe mechanisms can be applied to any Ramansystem design or setup (e.g., that in FIGS. 1, 3, 5, 9, 13-16, 20-28,30-40) disclosed herein. As another example, any of the scan patternsmay be executable by any Raman system design or setup disclosed herein.As yet another example, any Raman system disclosed herein can encompasstwo or more pump lasers as generally shown in FIG. 15.

CONCLUSION

While various inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize or be able toascertain, using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

Also, various inventive concepts may be embodied as one or more methods,of which an example has been provided. The acts performed as part of themethod may be ordered in any suitable way. Accordingly, embodiments maybe constructed in which acts are performed in an order different thanillustrated, which may include performing some acts simultaneously, eventhough shown as sequential acts in illustrative embodiments.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein, unless clearlyindicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein, should be understood to mean“either or both” of the elements so conjoined, i.e., elements that areconjunctively present in some cases and disjunctively present in othercases. Multiple elements listed with “and/or” should be construed in thesame fashion, i.e., “one or more” of the elements so conjoined. Otherelements may optionally be present other than the elements specificallyidentified by the “and/or” clause, whether related or unrelated to thoseelements specifically identified. Thus, as a non-limiting example, areference to “A and/or B”, when used in conjunction with open-endedlanguage such as “comprising” can refer, in one embodiment, to A only(optionally including elements other than B); in another embodiment, toB only (optionally including elements other than A); in yet anotherembodiment, to both A and B (optionally including other elements); etc.

As used herein, “or” should be understood to have the same meaning as“and/or” as defined above. For example, when separating items in a list,“or” or “and/or” shall be interpreted as being inclusive, i.e., theinclusion of at least one, but also including more than one, of a numberor list of elements, and, optionally, additional unlisted items. Onlyterms clearly indicated to the contrary, such as “only one of” or“exactly one of,” or, when used in any claims, “consisting of,” willrefer to the inclusion of exactly one element of a number or list ofelements. In general, the term “or” as used herein shall only beinterpreted as indicating exclusive alternatives (i.e., “one or theother but not both”) when preceded by terms of exclusivity, such as“either,” “one of,” “only one of,” or “exactly one of.” “Consistingessentially of,” when used in any claims, shall have its ordinarymeaning as used in the field of patent law.

As used herein, the phrase “at least one,” in reference to a list of oneor more elements, should be understood to mean at least one elementselected from any one or more of the elements in the list of elements,but not necessarily including at least one of each and every elementspecifically listed within the list of elements and not excluding anycombinations of elements in the list of elements. This definition alsoallows that elements may optionally be present other than the elementsspecifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elementsspecifically identified. Thus, as a non-limiting example, “at least oneof A and B” (or, equivalently, “at least one of A or B,” or,equivalently “at least one of A and/or B”) can refer, in one embodiment,to at least one, optionally including more than one, A, with no Bpresent (and optionally including elements other than B); in anotherembodiment, to at least one, optionally including more than one, B, withno A present (and optionally including elements other than A); in yetanother embodiment, to at least one, optionally including more than one,A, and at least one, optionally including more than one, B (andoptionally including other elements); etc.

As used herein, all transitional phrases such as “comprising,”“including,” “carrying,” “having,” “containing,” “involving,” “holding,”“composed of,” and the like are to be understood to be open-ended, i.e.,to mean including but not limited to. Only the transitional phrases“consisting of” and “consisting essentially of” shall be closed orsemi-closed transitional phrases, respectively, as set forth in theUnited States Patent Office Manual of Patent Examining Procedures,Section 2111.03.

The invention claimed is:
 1. A method of Raman spectroscopy, the methodcomprising: acquiring interleaved measurements of a first Raman signalof a sample using a Raman pump beam at a first wavelength, a secondRaman signal of the sample using a Raman pump beam at a secondwavelength different than the first wavelength, and ambient lighttransmitted and/or scattered by the sample; and generating apost-processed signature based on the interleaved measurements of thefirst Raman signal, the second Raman signal, and the ambient light,wherein acquiring the interleaved measurements comprises: measuring thefirst Raman signal over first measurement periods equal to a firstinteger multiple of a base period; measuring the second Raman signalover second measurement periods equal to a second integer multiple ofthe base period; and measuring the ambient light over third measurementperiods equal to a third integer multiple of the base period.
 2. Themethod of claim 1, wherein generating the post-processed signaturecomprises determining a difference between the first Raman signal andthe second Raman signal.
 3. The method of claim 1, wherein generatingthe post-processed signature comprises estimating a Raman signature ofthe sample and a fluorescent and ambient light background, based on thefirst Raman signal and the second Raman signal.
 4. The method of claim1, wherein measuring the first Raman signal comprises scanning a firstRaman pump beam through a scan pattern over at least a portion of thesample during each of the first measurement periods.
 5. The method ofclaim 1, wherein the first integer multiple of the base period is equalto the second integer multiple of the base period and the third integermultiple of the base period.
 6. The method of claim 1, wherein the baseperiod is based on a flicker period of an ambient light source.
 7. Themethod of claim 6, further comprising: determining the flicker period ofthe ambient light source based on a frequency analysis of the ambientlight intensity.
 8. The method of claim 6, wherein the base period is aninteger multiple of the flicker period.
 9. The method of claim 6,wherein the flicker period is an integer multiple of the base period.10. The method of claim 6, wherein the flicker period is about 8.33 msto about 10 MS.
 11. A system for Raman spectroscopy, the systemcomprising: at least one Raman pump source to emit a first Raman pumpbeam at a first wavelength and a second Raman pump beam at a secondwavelength different than the first wavelength; a detector to acquireinterleaved measurements of a first Raman signal of a sample in responseto the first Raman pump beam, a second Raman signal of the sample inresponse to the second Raman pump beam, and ambient light transmittedand/or scattered by the sample; and a processor, operably coupled to thedetector, to generate a post-processed signature based on theinterleaved measurements of the first Raman signal, the second Ramansignal, and the ambient light, wherein the detector is configured toacquire the interleaved measurements by: measuring the first Ramansignal over first measurement periods equal to a first integer multipleof a base period; measuring the second Raman signal over secondmeasurement periods equal to a second integer multiple of the baseperiod; and measuring the ambient light over third measurement periodsequal to a third integer multiple of the base period.
 12. The system ofclaim 11, further comprising: a beam scanner, in optical communicationwith the at least one Raman pump source, to scan the first Raman pumpbeam through a scan pattern over at least a portion of the sample duringeach of the first measurement periods.
 13. The system of claim 11,wherein the first integer multiple of the base period is equal to thesecond integer multiple of the base period and the third integermultiple of the base period.
 14. The system of claim 11, wherein thebase period is based on a flicker period of an ambient light source. 15.The system of claim 14, wherein the processor is configured to determinethe flicker period of the ambient light source based on a frequencyanalysis of the ambient light intensity.
 16. The system of claim 14,wherein the processor is configured to determine the flicker periodbased on an electric utility frequency at a location of the sample. 17.The system of claim 16, wherein the processor is configured to determinethe electric utility frequency based on the location of the sample. 18.A method of Raman spectroscopy of a sample, the method comprising:scanning a first Raman pump beam at a first wavelength in a scan patternacross the sample over a first scan period; measuring a first Ramansignal scattered by the sample in response to the first Raman pump beamover a first integration period; scanning a second Raman pump beam at asecond wavelength different than the first wavelength in the scanpattern across the sample over a second scan period; measuring a secondRaman signal scattered by the sample in response to the second Ramanpump beam over a second integration period; and forming a post-processedsignature based on the first Raman signal and the second Raman signal.19. The method of claim 18, wherein the sample is in a reaction vesselor a flow cell, and further comprising: identifying at least onechemical species in the reaction vessel based on the post-processedsignature.
 20. The method of claim 18, further comprising: measuringambient light transmitted and/or scattered by the sample over a thirdintegration period, and wherein forming the post-processed signature isbased on the first Raman signal, the second Raman signal, and theambient light.
 21. The method of claim 20, wherein the first integrationperiod is equal to the second integration period and the thirdintegration period.
 22. The method of claim 20, wherein the firstintegration period is based on a flicker period of the ambient light.23. The method of claim 22, wherein the first integration period isequal to an integer multiple of the flicker period.
 24. The method ofclaim 22, further comprising: determining the flicker period based on afrequency analysis of the measured ambient light intensity.
 25. Themethod of claim 22, further comprising: determining the flicker periodbased on an electric utility frequency at a location of the sample. 26.The method of claim 25, further comprising: determining the location ofthe sample based on a Global Positioning System (GPS) signal.
 27. Themethod of claim 25, further comprising: determining the location of thesample based on user input.